Methods of detecting oxidized calcium/calmodulin dependent protein kinase II

ABSTRACT

Calcium/calmodulin dependent protein kinase II (CaMKII) has been found to be directly oxidized, and direct oxidation of CaMKII was observed to result in calcium independent activation of CaMKII. Antibodies that bind specifically to oxidized forms of CaMKII (oxCaMKII) were generated and were utilized to detect oxCaMKII in blood from: (1) mice with cancer; (2) mice with a knock out of the gene encoding methionine sulfoxide reductase; (3) mice injected with angiotensin II; (4) mice injected with bacterial endotoxin; (5) mice fed a pro-oxidant (ketogenic) diet; and (6) mice with cancer that had been treated with experimental therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 12/430,644, filed on Apr. 27, 2009, whichapplication was published on Sep. 9, 2010, as US2010/0226929, and whichapplication claims the benefit under 35 U.S.C. §119(e) to U.S.Provisional Application No. 61/048,259, filed on Apr. 28, 2008, thecontents of which are incorporated herein by reference in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. Government Support from the followingagency: NIH, Grant Nos. R01 HL 079031, R01 HL 62494, and R01 HL 70250.The U.S. Government has certain rights in the invention.

FIELD

The field of the invention relates to calcium/calmodulin dependentprotein kinase II (CaMKII). In particular, the field relates to methodsfor detecting oxidized forms of CaMKII in order to characterize ordiagnose a disease or condition. The field also relates to methods formodulating the activity of CaMKII, either directly or indirectly, toorder to treat or prevent a disease or condition.

BACKGROUND

Excessive oxidation is thought to cause or contribute to heart failureand cardiac arrhythmias, cancer, premature aging, atherosclerosis,Alzheimer's disease, and sepsis. The multifunctional calcium andcalmodulin-dependent protein kinase II (CaMKII) now has been identifiedas a novel molecular target for activation by oxidation. (See Ericksone.g., accepted for publication in Cell, projected publication date May2, 2008 [hereinafter Erickson e.g., Cell 2008]).

Oxidation activates CaMKII (Erickson e.g., Cell 2008). Activated CaMKIIcan cause heart failure and arrhythmias and is implicated in progressionof cancer, neurological disease, and sepsis. In particular, CaMKIIoxidation has been shown to play a pivotal role in cardiac cell deathduring angiotensin II mediated apoptosis. CaMKII oxidation increasesduring myocardial ischemia and infarction (Erickson Cell 2008) andcauses cellular damage (Yang AJP 2006) and cardiac dysfunction aftermyocardial infarction (Zhang Nat Med 2005). Additionally, regulation ofCaMKII activity by oxidation may play a role in cancer, aging, sepsis,cell development and differentiation, because each of these conditionsis marked by increased oxidation and CaMKII activity.

Although oxidation is a bona fide pathological signal in important humandiseases, diagnostic measures of oxidative stress suitable for clinicalapplications are lacking. Another problem in utilizing alternativemeasures of oxidant stress is that they are not directly linked todisease progression. (See, e.g., Roberts Free Radical Biology andMedicine 2007, (discussing the molecular biology of isoprostanes).)Here, it is shown that oxidation of CaMKII causes enhanced CaMKIIactivity by preventing refolding of the enzyme into an inactive(resting) conformation. CaMKII is unusual because it is a target foroxidation and CaMKII's role in disease and the mechanism of activationby oxidation are understood. (See Erickson e.g., Cell 2008). Thus,measuring oxidized CaMKII may provide the first opportunity to trackoxidation of a biologically active molecule present in peripheral bloodor biopsy specimens, in contrast, other measures of oxidative stress are‘bystander’ molecules not directly implicated in disease pathogenesis(e.g., isoprostanes). In addition to diagnostic clinical applications,researchers in the areas of nerve, muscle, heart, infectious disease andcancer biology may benefit from a reagent that can be used to measureoxidant burden, and in particular oxidant-activated CaMKII.

Although CaMKII is a validated target for heart failure and arrhythmiasand is implicated as a causal agent in cancer, neurological diseases andaging, there are currently no available methodologies to non-invasivelymeasure oxidized CaMKII activity. A method to detect oxidized CaMKII maybe valuable as a diagnostic tool (e.g., to monitor the success ofantioxidant therapy, antibacterial therapy, chemotherapy or futuretherapies that involve inhibiting CaMKII). In addition, the ability tomeasure oxidized CaMKII may be invaluable to researchers attempting toconnect oxidative stress with CaMKII activation in these fields. Currentantibodies only detect either total CaMKII or the phosphorylated form ofCaMKII.

Here, an immune serum (rabbit) against the oxidized form of CaMKII wasdeveloped and purified by binding to protein A beads. This serum can beused to detect the presence of oxidized CaMKII in heart, blood and othertissues or body fluids using a number of techniques, including Westernblot and immunofluorescent imaging. This antiserum may be used tomeasure oxidized CaMKII in patients from peripheral blood and/or biopsyspecimens. In particular, the serum was utilized to detect elevatedlevels of oxidized CaMKII in blood from mice that serve as models forvarious human diseases or conditions.

SUMMARY

Calcium/calmodulin dependent protein kinase II (CaMKII) has been foundto be directly oxidized. Furthermore, direct oxidation of CaMKII wasobserved to result in calcium independent activation of CaMKII.Antibodies that bind specifically to oxidized forms of CaMKII (oxCaMKII)were generated and were utilized to detect oxCaMKII in blood from: (1)mice with cancer; (2) mice with premature aging due to knock out of thegene encoding methionine sulfoxide reductase A (MsrA−/−); (3) miceinjected with angiotensin II (a cause of cardiac hypertrophy andfailure); (4) mice injected with bacterial endotoxin (a model ofsepsis); (5) mice fed a pro-oxidant (ketogenic) diet; and (6) mice withcancer that had been treated with experimental therapy (e.g.,2-deoxyglucose or radiation).

Disclosed is an isolated or purified antibody or an antigen-bindingfragment thereof that binds specifically to oxidizedcalcium/calmodulin-dependent protein kinase II (oxCaMKII) or a fragmentthereof. The antibody may be monoclonal or polyclonal. In someembodiments, the antibody or antigen-binding fragment thereof is an Fabfragment, an F(ab′)₂ fragment, a dAb fragment, or a single chain Fv(scFv).

The antibody may include a human, mouse, rat, guinea pig, rabbit, dog,cat, pig, goat, horse or cow antibody, and in some embodiments, theantibody or antigen-binding fragment may be chimeric. In furtherembodiments, the antibody or antigen-binding fragment may be humanized.

Typically, the antibody or antigen-binding fragment thereof bindsspecifically to oxCaMKII or a fragment thereof, in some embodiments, theantibody or antigen-binding fragment thereof binds specifically to apeptide consisting of an amino acid sequence selected from the consensusamino acid sequence {S,C} {H,Q}RSTVAS{C,M}MHRQETV{D,E} (SEQ ID NO:4) inwhich the cysteine or methionine at position 9 and the methionine atposition 10 are oxidized. In further embodiments, the antibody orantigen-binding fragment thereof binds specifically to oxCaMKII deltahaving oxidized methionine residues at positions 281 and 282 (e.g., anoxidized peptide consisting of an amino acid sequence ofCQRSTVASMMHRQETVD (SEQ ID NO:5)).

The antibody or antigen-binding fragment thereof may include a labelsuch as a radiolabel, a fluorophore, an enzyme, a colloidal metal (e.g.,gold), or a colored nanoparticle, and the labeled antibody may beutilized in an immunoassay to detect oxCaMKII in a biological samplefrom a patient. Biological samples may include, but are not limited to,tissue samples and blood products (e.g., plasma).

The antibody or antigen-binding fragment thereof may be immobilized. Forexample, the antibody or antigen-binding fragment thereof may becovalently or non-covalently bound to a solid support. The immobilizedantibody or antigen-binding fragment thereof may be utilized in animmunoassay to detect oxCaMKII in a biological sample from a patient.

Also disclosed is an isolated or purified CaMKII polypeptide or animmunogenic fragment thereof that include one or more oxidizedmethionine residues at amino acid positions 281 or 282 (preferably bothmethionine residues at amino acid positions 281 and 282). Theimmunogenic fragment preferably includes at least about 8 amino acids(more preferably at least about 10, 12, 14, 16, 18, or 20 amino acids)that span amino acid positions 281 or 282 (e.g., an immunogenic fragmentthat includes amino acid sequences of SEQ ID NOS:1, 2, 3, 4, or 5).

Also disclosed are compositions that comprise the isolated or purifiedCaMKII polypeptide or an immunogenic fragment thereof that include oneor more oxidized methionine residues at amino acid positions 281 or 282(i.e., oxCaMKII). In some embodiments of the compositions, oxCaMKIIrepresents at least about 50% of the total amount of CaMKII in thecomposition (preferably oxCaMKII represents at least about 70% of thetotal amount of CaMKII in the composition, more preferably oxCaMKIIrepresents at least about 90% of the total amount of CaMKII in thecomposition).

Also disclosed is a method for preparing antisera that bindsspecifically to oxidized calcium/calmodulin-dependent protein kinase II(oxCaMKII). The method typically includes: (a) administering to ananimal a composition comprising CaMKII or an immunogenic fragmentthereof having one or more oxidized amino acids selected from a groupconsisting of oxidized methionine and oxidized cysteine; (b) andisolating antisera from the animal. Optionally, the composition furtherincludes an adjuvant. The oxCaMKII or the immunogenic fragment thereofmay be prepared by reacting CaMKII or an immunogenic fragment thereofwith an oxidizing agent. Preferably, the one or more oxidized aminoacids include oxidized methionine residues at positions 281 and 282relative to the full-length CaMKII delta polypeptide or at correspondingpositions in an Immunogenic fragment of CaMKII delta polypeptide (e.g.,oxidized methionine residues at amino acid positions 9 and 10 of SEQ IDNO:5).

Also disclosed are isolated or purified cells that produce antibody thatbinds specifically to oxCaMKII or an oxidized fragment thereof. Cellsthat produce antibody may include hybridomas or plasmacytomas.

The disclosed antibody or antigen binding fragment thereof may beutilized in methods for detecting oxCaMKII or fragments thereof. Forexample, a method of detecting oxCaMKII may include: (a) contacting abiological sample from a patient with an antibody or antigen-bindingfragment thereof that binds specifically to oxCaMKII and forms acomplex; and (b) detecting the complex. Suitable biological samples mayinclude, but are not limited to, blood and plasma. The method may beutilized to characterize a disease or condition in the patient (e.g., acardiac disease or condition, cancer, premature aging, atherosclerosis,Alzheimer's disease, or sepsis).

In one embodiment, the disclosed methods are utilized to detect oxCaMKIIin a biological sample from a patient where the patient has or is atrisk for developing a disease or condition selected from a groupconsisting of a cardiac disease, cancer, premature aging,atherosclerosis, Alzheimer's disease, or sepsis. The method further mayinclude: (c) administering a therapeutic agent to the patient (or notadministering a therapeutic agent to the patient) based on detectingoxCaMKII (or not detecting oxCaMKII) in the biological sample from thepatient.

In a further embodiment, the method may be utilized to detect oxCaMKIIin a patient that is undergoing pharmacological therapy (e.g.,pharmacological therapy with an angiotensin converting enzyme (ACE)inhibitor, an antioxidant agent, an antibacterial agent, or ananticancer agent). In this further embodiment, the method further mayinclude: (c) modulating the pharmacological therapy based on detectingor not detecting oxCaMKII. For example, modulating the therapy mayinclude increasing or decreasing dosage of the pharmacological agentbased on detecting or not detecting oxCaMKII. The methods may includeassessing oxidative stress.

Also disclosed are kits for detecting oxCaMKII. The kits may include:(a) an antibody or antigen-binding fragment thereof that bindsspecifically to oxCaMKII to form a complex; and (b) a label fordetecting the complex.

Further disclosed are pharmaceutical compositions. The compositions mayinclude: (a) an antibody or antigen-binding fragment thereof that bindsspecifically to oxCaMKII; and (b) a pharmaceutically acceptable carrier.

Further disclosed are methods for treating or preventing structuralheart disease in a patient. The methods include administering to thepatient an antibody or antigen-binding fragment thereof that bindsspecifically to oxCaMKII, for example where the antibody orantigen-binding fragment inhibits activity of oxCaMKII. In someembodiments of the methods, the antibody or antigen-binding fragmentbinds specifically to oxCaMKII delta having oxidized methionine residuesat positions 281 and 282.

Further disclosed are methods for treating or preventing structuralheart disease in a patient, which methods include administering to thepatient a therapeutic agent that specifically inhibits oxidation ofCaMKII. For example, the therapeutic agent may specifically inhibitoxidation of CaMKII at methionine residues present at amino acidpositions 281 and 282. In some embodiments, the methods includeadministering to the patient a therapeutic agent that increasesmethionine sulfoxide reductase (MSR) activity in the patient. Thetherapeutic agent may increase MSR activity (e.g., by increasing MSRexpression) which subsequently augments the conversion of oxidizedmethionines in CaMKII to non-oxidized methionines (e.g., augmentingconversion of oxidized methionine residues present at amino acidpositions 281 and 282 of CaMKII to non-oxidized states).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. CaMKII is activated by reactive oxygen species (ROS). (A)General structure of a subunit from the multimeric holoenzyme CaMKII andmechanism of CaMKII activation by autophosphorylation. The amino acidsequence of the regulatory domain is highlighted to show theautoinhibitory (AI) and calmodulin-binding (CaM-B) regions. Yellowsymbols represent CaM. Pretreatment with Ca²⁺/CaM (1°) followed byphosphorylation at T287 (2°) yields persistent activity even after theremoval of Ca²⁺/CaM (3°). (B) Kinase assays were performed after threedistinct treatment steps: (1°) Ca²⁺/CaM, (2°)±H₂O₂ or ATP, and(3°)±EGTA. (n=6 assays/group, * p<0.05 vs. WT no treatment). (C) CaMKIIis activated by H₂O₂ in a dose-dependent manner after pre-treatment withCa²⁺/CaM. Oxidation-dependent CaMKII activity is ablated in M281/282Vmutants (n=6 assays/group, * p<0.05 vs. WT no treatment). (D) M281/282Vmutants have normal Ca²⁺/CaM-dependent andT287-autophosphorylation-dependent activation (n=6 assays/group, *p<0.05 vs. WT no treatment). (E) Proposed mechanism for activation ofCaMKII by oxidation. After initial activation of the holoenzyme byCa²⁺/CaM (1°), oxidation at M281/282 (2°) blocks re-association of thecatalytic domain, yielding persistent CaMKII activity (3°).

FIG. 2. Oxidation and autophosphorylation of CaMKII are associated withsimilar fluorescence shifts. (A) Sample emission fluorescence spectra ofWT CaMKII after no treatment or in the presence of Ca²⁺/CaM, ATP, orH₂O₂. No significant difference among CaMKII mutants for peakfluorescence with no treatment was observed (n=3 assays/group, notshown). (S) M28.1/282V CaMKII mutants do not show a shift in emissionfluorescence intensity at peak fluorescence after treatment with 100 μMH₂O₂ (n=3 assays/group, * p<0.05 vs. WT no treatment). Response toCa²⁺/CaM and ATP activation are unchanged.

FIG. 3. ROS-dependent activity is ablated by M281V or M282V pointmutation. Wild type CaMKII shows increased activity in response to H₂O₂treatment, while this dose dependent activation is not seen in the M281Vand M282V mutants. (n=3 assays/group, * p<0.05 vs. WT no treatment)

FIG. 4. Oxidation of CaMKII does not initiate CaM trapping duringROS-dependent CaMKII activation. (A) Real time fluorescence anisotropymeasurement of CaM after the addition of CaCl₂, CaMKII, and EGTA (blackline). Addition of H₂O₂ prior to EGTA (red line) does not result in CaMtrapping. CaM trapping is seen when ATP is present (blue and greenlines). (B) In contrast to WT CaMKII, an M308V mutant shows slowedCa²⁺/CaM/CaMKII dissociation after treatment with H₂O₂. (C) Half time tobaseline fluorescence after addition of EGTA for Ca²⁺/CaM/CaMKIIdissociation, with WT or M308V CaMKII (n=3 trials/group, * p<0.05 vs.half time for CaMKII in buffer only). (D) Pretreatment of CaMKII withiodoacetic acid blocks cysteine oxidation but does not affectROS-dependent activity. Mutation of C290 does not affect CaMKII activityresponses to H₂O₂.

FIG. 5. AngII induces oxidation of CaMII in vivo. (A) Immunoblot of WTCaMKII and M281/282V mutant after no treatment, oxidation, orautophosphorylation probed with antibodies against total,autophosphorylated (p-T287), or oxidized CaMKII. Summary data showsrelative band intensity using the oxidized CaMKII antibody (n=3trials/group, * p<0.05 vs. band intensity of WT CaMKII treated withH₂O₂). (B) Immunoblot and summary data of oxidized WT CaMKII probed withantiserum against oxidized M28.1/282 with increasing ratios of oxidizedantigen peptide. (n=3 trials/group, * p<0.05 vs. band intensity with nopeptide). (C) Immunofluorescent staining of heart sections from micetreated with saline, AngII, or Iso and probed for oxidized or totalCaMKII. Red staining is positive for oxidized or total CaMKII and bluestaining is for nuclei. Calibration bars are 100 microns. (D) Immunoblotand summary data of heart lysates from mice treated with saline (Sal),Iso, or AngII probed with antibodies against total CaMKII, oxidizedCaMKII, or actin (n=3 hearts/group, * p<0.05 vs. band intensity ofsaline treatment).

FIG. 6. Wild type heart sections have increased T287-phosphorylatedCaMKII after AngII treatment. Wild type and p47^(−/−) mice were treatedwith AngII (3 mg/kg/day) for 1 week. Heart sections from these mice werestained with an antibody against T287-phosphorylated or total CaMKII.

FIG. 7. AngII increases ROS production and apoptosis by aCaMKII-dependent pathway in cardiomyocytes. (A) Percent of totalisolated, cardiomyocytes positive for TUNEL staining after treatmentwith saline, AngII, Iso, or H₂O₂ (n=6 hearts/group, * p<0.05 vs. WT withsaline). (B) Caspase-3 activity induced by saline, AngII, or Isonormalized to WT cells treated with saline (n=hearts/group, * p<0.05 vs.WT with saline). (C) DHE stained cardiomyocytes after treatment with 100nM AngII or Iso. Red coloration indicates presence of ROS above controlcells. Scale bars equal 50 μm. (D) Percent of total cells positive forDHE staining above control (n=3 assays/group. * p<0.05 vs. WT saline).(E) Example traces of intracellular calcium concentration of cultured WTcardiomyocytes treated with 100 nM AngII (red symbols) or Iso (bluesymbols) measured by real-time calcium imaging. The arrow indicatesaddition of AngII or Iso. (F) Peak intracellular Ca²⁺ concentration inresponse to either AngII or Iso for WT or p47^(−/−) cells (n=3trials/group, NS=not statistically different).

FIG. 8. AngII-induced apoptosis is blocked by CaMKII silencing. (A)Representative immunoblot with anti-CaMKII to measure protein expressionafter treatment with shRNA and shRNA-resistant rescue constructs.Immunoblot against actin was used as a loading control (not shown).Middle panel shows summary data of CaMKII expression relative tountreated cells (n=3 experiments/group, * p<0.05 vs. no treatment).Bottom panel shows summary data for CaMKII activity assays of lysates(n=3 experiments/group, * p<0.05 vs. total activity with no treatment, †p<0.05 vs. ROS-dependent activity with no treatment). Only the WT CaMKIIconstruct was able to reconstitute both Ca²⁺/CaM- and ROS-dependentactivity observed in untreated cells. (B) Immunostaining and (C) summarydata from isolated rat cardiomyocytes transduced with shRNA againstCaMKII followed by rescue with WT CaMKII, M281/282V, or GFP control.Immunostaining shows total nuclei (DAPI) and DMA nicking (TUNEL)consistent with apoptosis. Scale bars equal 100 μm. Summary data showpercent of total nuclei with positive TUNEL staining (n=6hearts/group, * p<0.05 vs. GFP with AngII).

FIG. 9. AngII causes cardiac apoptosis in vivo via a ROS andCaMKII-mediated pathway. (A) Immunostaining of mouse heart sections fortotal nuclei (DAPI) and nuclear damage (TUNEL) consistent withapoptosis. WT, p47^(−/−), and AC3-I mice were treated with Ang II (3mg/kg/day) or Iso (30 mg/kg/day) for seven days. Scale bars equal 100μm. (B) Percent of total nuclei that showed positive TUNEL staining (n=3hearts/group, * p<0.05 vs. WT with saline).

FIG. 10. MsrA^(−/−) mice have increased susceptibility to AngII-mediatedapoptosis. (A) Immunofluorescent staining of heart sections from WT andMsrA^(−/−) mice treated with AngII and probed for oxidized or totalCaMKII. Red stamina is positive for oxidized or total CaMKII and bluestaining is for nuclei. Calibration bars are 100 microns. (B)Quantification of average staining intensity for AngII treated hearts,relative to WT (n=3 hearts/group, * p<0.05 vs. WT with AngII). (C)Summary data for TUNEL staining of heart sections from WT and MsrA^(−/−)mice treated with saline or AngII (n=5 hearts/group, * p<0.05 vs. WTwith saline).

FIG. 11. Mice lacking MsrA have increased CaMKII oxidation, apoptosis,reduced survival and impaired heart function after myocardialinfarction. (A) Immunostaining and (B) stain intensity quantification ofoxidized CaMKII in heart sections from WT, p47^(−/−), and MsrA^(−/−)mice post-MI (n=3 hearts/group, * p<0.05 vs. WT). (C) Summary data forTUNEL staining of heart sections from WT, p47^(−/−), and MsrA^(−/−) micepost-MI (n=3 hearts/group, * p<0.05 vs. WT). (D) Mortality issignificantly increased post-MI in MsrA^(−/−) mice compared to WTcontrols. Numbers in bars represent post-MI deaths/total number of micereceiving MI. Post-MI left ventricular dilation (E) and function (F)were compromised in surviving MsrA^(−/−) mice compared to WT controlsthree weeks after surgery (n=17 hearts/group for WT, n=9 hearts/groupfor MsrA^(−/−)).

FIG. 12. The identity of cultured neonatal mouse cardiomyocytes isconfirmed by confocal microscopy after immunostaining. Green stain isα-actinin and purple stain is nuclei. Over 90% of nuclei fall clearlywithin striated cardiomyocytes. Calibration bars are 20 μm.

FIG. 13. The identity of cardiomyocytes from mouse heart, sections isconfirmed by confocal microscopy after immunostaining. (A) Green stainis α-actinin and blue stain is nuclei. (B) Green stain is α-actinin andred stain is TUNEL. Over 90% of nuclei fall clearly within striatedcardiomyocytes. Calibration bars are 20 μm.

FIG. 14. A. Immunodetection of oxidized CaMKII (by H₂O₂) using antiserum(lower panel marked ‘oxidized’). B. Immunodetection of oxidized CaMKIIin heart in vivo after AngII infusion (right panel, red staining)compared to saline (left panel). Blue dapi staining indicates nuclei inboth panels.

FIG. 15. Angiotensin II (AngII) and endotoxin (Endo) treatment resultsin increased oxidized CaMKII (oxCaMKII) in blood. Wild type (WT) andMsrA−/− mice were treated with saline, AngII, or Endo and sacrificed 6hours later. The top (oxidized CaMKII) and bottom (total CaMKII) blotsare of paired samples. From left to right: wild type mice treated withsaline injection (lane 1); wild type mice treated with angiotensin II tomimic signaling in myocardial hypertrophy and heart failure (lane 2);wild type mice treated with bacterial endotoxin to mimic sepsis (lane3); MsrA knock out mice—a model of premature aging—treated with saline(lane 4); MsrA knock out mice treated with angiotensin II—a model ofheart disease as part of premature aging (lane 5); MsrA knock out micetreated with bacterial endotoxin—a model of sepsis in aging (lane 6).Western blot of blood samples and ImageJ analysis were used to quantifytotal and oxidized CaMKII. Summary data show the band intensity ofoxCaMKII relative to WT treated with saline, normalized for total CaMKIIpresent. N=1 for each group.

FIG. 16. Cancer and w-deoxyglucose (2DG) treatment are associated withconcomitant increases in oxCaMKII in blood. Mice were exposed to cancercells and treated with either saline or 2DG. Those mice that failed todevelop tumors after 60 days were labeled as controls. The top (oxidizedCaMKII) and bottom (total CaMKII) blots are of paired samples. From leftto right: healthy control mice (lanes 1 and 2); increased oxidizedCaMKII in a mouse with cancer (lane 3); mice with cancer treated with2-deoxyglucose—an experimental cancer therapy—show markedly increasedoxidized CaMKII (lanes 4 and 5), Western blot of blood samples andImageJ analysis were used to quantify total and oxidized CaMKII. Summarydata show the band intensity of oxCaMKII relative to Control 1,normalized for total CaMKII present. N=1 for each group.

FIG. 17. A sample experiment to probe for the presence of oxCaMKII inmice bearing cancer and post-treatment. Mice with no cancer, FADU (headand neck cancer), or human lung cancer were treated with saline, 2DG,radiation (rad), or given a ketogenic diet (keto). The table indicatescancer type and treatment for each sample.

FIG. 18. Both cancer and varied treatments increase blood oxCaMKII in anadditive manner. Mice were divided into four groups based on thepresence or absence of cancer and whether or not they receivedtreatment. Western blot of blood samples and ImageJ analysis were usedto quantify total and oxidized CaMKII. Summary data show the mean bandintensity of oxCaMKII relative to negative cancer/negative treatment, inall cases normalized for total CaMKII present. N values for each groupare indicated. Error bars represent SEM.

DETAILED DESCRIPTION Definitions

The present invention is described herein using several definitions, asset forth below and throughout the application.

As used herein, “a,” “an,” and “the” mean “one or more” unless thecontext clearly dictates otherwise. For example, reference to “anantibody” includes one or more antibodies. Reference to “an inhibitor”includes one or more inhibitors.

As used herein, “about,” “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of the term which are not clear to persons of ordinaryskill in the art given the context in which it is used, “about” or“approximately” will mean up to plus or minus 10% of the particular termand “substantially” and “significantly” will mean more than plus orminus 10% of the particular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising.”

As used herein, a “patient” may be interchangeable with “subject” andincludes human and non-human animals. Non-human animals may includedogs, cats, horses, cows, pigs, sheep, and the like.

A “patient in need thereof” may include a patient in need of diagnosis,characterization, treatment, prognosis, or prevention with respect to adisease or condition associated with oxidation of calcium, calmodulindependent protein kinase II. Examples of diseases or conditions mayinclude, but are not limited to, cardiac diseases or conditions, cancer,premature aging, atherosclerosis, Alzheimer's disease, and sepsis. A“patient in need thereof” may include a patient undergoing therapy totreat a disease or condition that may include, but is not limited to, acardiac disease or condition, cancer, premature aging, atherosclerosis,Alzheimer's disease, and sepsis. For example, a “patient in needthereof” may include a patient having heart failure, where the patientis undergoing a pharmacological treatment to reduce cardiac dysfunctionor mortality (e.g., treatment with angiotensin-converting enzyme (ACE)inhibitors). Furthermore, a “patient in need thereof” may include apatient having a cardiac disease or condition, cancer, premature aging,atherosclerosis, Alzheimer's disease, or sepsis, where the patient isundergoing a pharmacological therapy selected from an anti-oxidanttherapy, an anti-bacterial therapy, or an anti-cancer therapy.

As used herein, “cardiac diseases or conditions” may include structuralheart diseases (e.g., myocardial infarction, cardiac dysfunctionfollowing myocardial infarction, reduced myocardial contractility,end-stage valve disease, and dilated cardiomyopathy). Cardiac diseasesor conditions may include those diseases or conditions associated withischemic injury, which means the damage or potential damage to an organor tissue that results from the interruption of blood flow to the organor tissue (i.e. an “ischemic event”). A “patient in need thereof” can bea patient diagnosed as having a myocardial infarction. The subject canbe a patient diagnosed as having post-infection cardiac dysfunction. Thesubject can be a patient who has been diagnosed as having had amyocardial infarction who is, thus, at increased risk of developingpost-infarction cardiac dysfunction. Furthermore, the subject can be apatient diagnosed as having dilated cardiomyopathy or symptoms of heartfailure from any cause associated with a phenotype of cardiac chamberdilation and reduced myocardial contractile function. The subject can bea patient diagnosed as having reduced myocardial contractility. Thesubject can be a patient diagnosed with atrial fibrillation.

As used herein, “CaMKII” refers to the enzyme “calcium/calmodulindependent protein kinase II.” In humans, there are four separate, highlyhomologous genes for CaMKII called alpha, beta, delta, or gamma (or α,β, δ and γ). Multiple isoforms of these genes are expressed throughalternative splicing mechanisms. Representative sequences for theisoforms of these genes have been submitted to public depositories suchas GenBank and include: GenBank Accession No, NP_(—)741960, CaMKII alphaisoform 2; GenBank Accession No. NP_(—)057065, CaMKII alpha isoform 1;GenBank Accession No. NP_(—)742079, CaMKII beta isoform 6; GenBankAccession No. NP_(—)742080, CaMKII beta isoform 7; GenBank Accession No.NP_(—)742077, CaMKII beta isoform 4; GenBank Accession No. NP_(—)001211,CaMKII beta isoform 1; GenBank Accession No. NP_(—)742081, CaMKII betaisoform 8; GenBank Accession No. NP_(—)742078, CaMKII beta isoform 5;GenBank Accession No, NP_(—)742076, CaMKII beta isoform 3; GenBankAccession No. NP_(—)742075, CaMKII beta isoform 2; GenBank Accession No.NP 001212, CaMKII delta isoform 3; GenBank Accession No. NP_(—)742126,CaMKII delta isoform 2; GenBank Accession No. NP_(—)742125, CaMKIIisoform 1; GenBank Accession No. NP 742113, CaMKII isoform 1; GenBankAccession No. NP_(—)001020609, CaMKII delta isoform 2 (SEQ ID NO: 12);NP_(—)751910, CaMKII gamma isoform 3; GenBank Accession No.NP_(—)751913, CaMKII gamma isoform 6; GenBank Accession No.NP_(—)751913, CaMKII gamma isoform 6; GenBank Accession No.NP_(—)7519.11, CaMKII gamma isoform 1; GenBank Accession No.NP_(—)751909, CaMKII gamma isoform 2; GenBank Accession No.NP_(—)751909, CaMKII gamma isoform 2; GenBank Accession No.NP_(—)001213, CaMKII gamma isoform 4; all of which GenBank entries areincorporated herein by reference in their entireties.

CaMKIIα and β are neuronal, while CaMKIIδ and γ are expressed in neuronsand in peripheral (non-neuronal) tissue. In some embodiments, thedisclosed antiserum detects oxidation of a Met pair at positions 281/282in CaMKIIδ, the predominant peripheral form of CaMKII. Based on the factthat this Met pair is conserved in CaMKIIβ and γ, it is expected thatthe disclosed antiserum will detect the oxidized forms of these otherisoforms. In CaMKIIα, which is a neuronally expressed CaMKII, a Cys issubstituted for the first Met at position 280 (corresponding to Metposition 281 in CaMKIIβ, δ and γ). However, it is known, that CaMKIIα isactivated by oxidation with a similar H₂O₂ dose-activity relationshipcompared to CaMKIIδ and that mutation of Met281Cys in CaMKIIδ (to mimicthe Cys-Met in CaMKIIα at positions 280/281) does not affect oxidativeactivation. Therefore, all of the four CaMKII isoforms are activated byoxidation via a similar mechanism (i.e., oxidation of Mets 281/282 in β,γ and δ or oxidation of Cys280 and Met281 in α). Because the Cys sidechain is a well-recognized target for oxidation, it is expected that thedisclosed antisera will be effective against each of the CaMKII isoforms(α-γ).

The CaMKII polypeptides disclosed herein may include the consensussequence {S,C} {H,Q}RSTVAS{C,M}MHRQETV{D,E} (SEQ ID NO:4). For example,full-length CaMKII polypeptides may include the consensus sequence {S,C}{H,Q}RSTVAS{C,M}MHRQETV{D,E} (SEQ ID NO:4) at about amino acid positions272-288 in a full-length isoform of CaMKII alpha or at about amino acidpositions 273-289 in a full-length isoform of CaMKII beta, delta, orgamma. The CaMKII polypeptides disclosed herein may comprise an oxidizedcysteine or methionine residue at positions 9 and 10 of the consensussequence {S,C} {H,Q}RSTVAS{C,M}MHRQETV{D,E} (SEQ ID NO:4), whichoxidized residue or methionine residue may be present at about aminoacid positions 280 and 281 in a full-length isoform of CaMKII alpha orat about amino acid positions 281 and 282 in a full-length isoform ofCaMKII beta, delta, or gamma.

Disclosed herein are antibodies or antigen binding fragments thereofthat bind specifically to an oxidized form of CaMKII or a fragmentthereof. The term “antibody” as used herein refers to an immunoglobulinmolecule or an immunologically active portion thereof (i.e., anantigen-binding portion). As used herein, the term “antibody” refers toa protein comprising at least one, and preferably two, heavy (H) chainvariable regions (abbreviated as VH), and at least one and preferablytwo light (L) chain variable regions (abbreviated as VL). The VH and VLregions can be further subdivided into regions of hypervariability,termed “complementarity determining regions” (“CDR”), interspersed withregions that are more conserved, termed “framework regions” (FR). Theextent of the framework region and CDR's has been precisely defined.(See, e.g., Kabat, E. A., e.g. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No, 91 3242; and Chothia, C. e.g. (1987)J. Mol. Biol. 196:901-917; which are incorporated herein by reference).Each VH and VL is composed of three CDR's and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4.

The term “antigen-binding fragment” of an antibody (or simply “antibodyportion” or “fragment”), as used herein, refers to one or more fragmentsof a full-length antibody that retain the ability to specifically bindto the antigen (e.g., oxidized CaMKII or a fragment thereof). Examplesof antigen-binding fragments of the disclosed antibodies include, butare not limited to: (i) an Fab fragment or a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains; (ii) an F(ab′)₂ fragmentor a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) an Fd fragment consisting ofthe VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VIIdomains of a single arm of an antibody, (v) a dAb fragment (Ward e.g.,(1989) Nature 341:544 546), which consists of a VH domain; and (vi) anisolated complementarity determining region (CDR). Even though the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv or “scFv,” (See, e.g., Bird e.g. (1988) Science 242:423 426; andHuston e.g. (1988) Proc. Natl. Acad. Sci. USA 85:5879 5883). Singlechain Fv or “scFv” are encompassed within the term “antigen-bindingfragment” of an antibody.

The disclosed antibodies can be full-length (e.g., an IgG (e.g., anIgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1, IgA2), IgD, and IgE) orcan include only an antigen-binding fragment (e.g., a Fab, F(ab′)₂ orscFV fragment, or one or more CDRs). The antibodies disclosed herein maybe a polyclonal or monoclonal antibodies. The disclosed antibodies maybe monospecific, (e.g., a monoclonal antibody, or an antigen-bindingfragment thereof), or may be multispecific (e.g., bispecific recombinantdiabodies). In some embodiments, the antibody can be recombinantlyproduced (e.g., produced by phage display or by combinatorial methods).In some embodiments, the antibodies (or fragments thereof) arerecombinant or modified antibodies (e.g., a chimeric, a humanized, adeimmunized, or an in vitro generated, antibody).

The disclosed antibodies or antigen binding fragments thereof bindspecifically to oxidized CaMKII or an oxidized fragment thereof (e.g.,an oxidized peptide comprising an amino acid sequence of SEQ ID NO:1, 2,3, 4, or 5). As disclosed herein, an antibody that binds specifically tooxidized CaMKII will bind to oxidized CaMKII and will not bind tonon-oxidized CaMKII. For example, an antibody that binds to CaMKII(beta, delta, or gamma) having oxidized methionine residues at positions281/282 and that does not bind to CaMKII (beta, delta, or gamma) havingnon-oxidized methionine residues at positions 281/282 is an antibodythat binds specifically to oxidized CaMKII. An antibody that binds toCaMKII alpha having oxidized cysteine and methionine residues atpositions 280/281 and that does not bind to CaMKII alpha havingnon-oxidized cysteine and methionine residues at positions 280/281 is anantibody that binds specifically to oxidized CaMKII.

The disclosed antibodies or antigen binding fragments thereof mayinclude a label. Labels may include, but are not limited toradioisotopes, bioluminescent compounds, chemiluminescent compounds,fluorescent compounds, metal chelates, an enzymes, colloidal metals(e.g., gold), and colored nanoparticles.

The presently disclosed methods may include performing immunoassays thatutilize an antibody or antigen binding fragment thereof againstoxCaMKII. The term “immunoassay” as used herein refers to a method ofdetecting or measuring antigens, in this case oxCaMKII, by usingantibodies or antigen binding fragments thereof as reagents. Theantibodies can be polyclonal, or, preferably, monoclonal. The terms“polyclonal antibodies” and “monoclonal antibodies” have the standardmeanings understood by those skilled in the art and refer to antibodies,either a mixture of different antibodies in the case of polyclonalantibodies, or a single antibody in the case of monoclonal antibodies,both of which are produced, in general, by immunization of an animalwith an antigen, in the case of monoclonal antibodies,antibody-producing cells are selected from the animal and fused withmyeloma cells. These cells are then cultured. The antibodies of thepresent invention detect oxCaMKII to a desired level. Harlow, E. andLane, D. (1988) Antibodies: A Laboratory Manual (Cold Spring HarborLaboratory), which is incorporated by reference in its entirety hereinteaches methods regarding the making and usage of antibodies. Theanti-oxCaMKII antibodies and antigen binding fragments thereof may belabeled or immobilized as disclosed herein.

In the disclosed immunoassays, the antibodies or antigen bindingfragments thereof may be utilized in liquid phase or bound to a solidphase carrier. Examples of types of immunoassays include competitive andnon-competitive immunoassays in either a direct or indirect format.Further examples of such immunoassays are the radioimmunoassay (RIA) andthe sandwich (immunometric) assay. Detection of the antigens using themonoclonal antibodies of the invention can be performed utilizingimmunoassays which are run in either the forward, reverse, orsimultaneous modes, including immunohistochemical assays onphysiological samples. Alternatively, the antibody of the invention canbe used to detect oxidized forms of CaMKII present inelectrophoretically dispersed gel protocols such as Western blots andtwo-dimensional gels. The antibodies or antigen binding fragmentsthereof may be bound to carriers and used to detect the presence ofoxidized forms of CaMKII. Examples of well-known carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, agaroses andmagnetite.

The presently disclosed methods and immunoassays may include determininga control value for oxCaMKII. The terra “control value” as used hereinrefers to a basal level of oxCaMKII that is normal (i.e., the amountpresent in a corresponding healthy cohort in the absence of anypathology (disease or disorder) which is associated with oxCaMKII). Suchcontrol values may account for the age of the individual and may bedirected to certain age ranges. Such control values additionally mayaccount for gender, race, and environmental exposures to pro-oxidants(e.g., smoking, diet, and the like).

Also disclosed are oxidized forms of CaMKII or immunogenic fragmentsthereof. The oxidized forms of CaMKII or immunogenic fragments thereofmay be isolated or purified (e.g., where an isolated or purified,oxidized form of CaMKII or an immunogenic fragment thereof represents atleast about 50% of total protein in an isolated or purified sample, andpreferably at least about 70%, 90%, or 95% of total protein in anisolated or purified sample). In some embodiments, the oxidized forms ofCaMKII or immunogenic fragments include at least one oxidized methionineor at least one oxidized cysteine. An oxidized methionine may bepartially or fully oxidized and may include methionine sulfoxide andmethionine sulfone. An oxidized cysteine may be partially or fullyoxidized and may include cystine and cysteic acid. An oxidized,immunogenic fragment of CaMKII typically includes at least about 8 aminoacids. In some embodiments, an oxidized, immunogenic fragment of CaMKIIcomprises or consists of an amino acid sequence selected from theconsensus sequences STVAS{C,M}MHR (SEQ ID NO:2), TVAS{C,M}MHRQE (SEQ IDNO:3) or {S,C} {H,Q}RSTVAS{C,M}MHRQETV{D,E} (SEQ ID NO:4) and may beformulated as an immunogenic or pharmaceutical composition together witha carrier and optionally an adjuvant.

An oxidized, immunogenic fragment of CaMKII may be used in a method forpreparing antibodies or antigenic binding fragments thereof that bindspecifically to oxidized CaMKII or an oxidized fragment thereof. Forexample, an oxidized, immunogenic fragment of CaMKII may be administeredto a host animal (e.g., together with an adjuvant) to generate anantibody response. Sera may be collected from the host, animal, orantibody producing cells may be isolated from the immunized animal(e.g., spleen cells) to generate immortalized antibody producing cells(e.g., hybridomas or plasmacytomas). Accordingly, hybridomas orplasmacytomas that produce monoclonal antibodies that specifically bindto oxidized CaMKII or an oxidized fragment thereof are contemplatedherein. Suitable host animals may include, but are not limited to, mice,rats, rabbits, and the like.

Oxidized forms of CaMKII or immunogenic fragments thereof may beprepared by reacting the CaMKII or Immunogenic fragment thereof with anoxidizing agent that converts methionine to methionine sulfoxide ormethionine sulfone under physiological conditions. An oxidizing agentalso may convert cysteine to cystine or cysteic acid under physiologicalconditions. Oxidizing agents may include, but are not limited tohydrogen peroxide (H₂O₂), alkyl peroxide, peroxy acids, ozone (O₃),polyatomic oxygen O₇, polyatomic oxygen O₈, NaIO₄, and potassiumperoxymonosulfate (oxone) (Wozniak e.g., Bioorg. Med. Chem. Lett.,8(19):2641 6 (1998)).

Also disclosed herein are mutant or variant forms of CaMKII andpolynucleotides that encode mutant or variant forms of CaMKII. In someembodiments, a mutant CaMKII includes one or more amino acidsubstitutions with respect to the amino acid sequence of wild-typeCaMKII. For example, a mutant CaMKII may include an amino acidsubstitution of a sulfur containing amino acid (e.g., a methionine or acysteine) for a non-sulfur containing amino acid. Non-methionine andnon-cysteine amino acids may include, but are not limited to, glycine,alanine, valine, leucine, isoleucine, phenylalanine, proline, serine,threonine, tyrosine, asparagine, glutamine, tryptophan, aspartic acid,glutamic acid, lysine, arginine, and histidine. In some embodiments, amutant CaMKII includes one or more substitutions of methionine for anon-methionine residue (e.g., MetMet→ValVal at positions 281/282 inCaMKII beta, delta, or gamma, as indicated in the polypeptide of SEQ IDNO: 13). In other embodiments, a mutant CaMKII includes one or moresubstitutions of cysteine and methionine for non-cysteine/non-methionineresidues (e.g., CysMet→ValVal at positions 280/281 in CaMKII alpha).

In some embodiments, the disclosed antibodies or antigen bindingfragments thereof, polypeptides, or polynucleotides (collectively“compounds”) may be formulated as pharmaceutical compositions thatinclude a therapeutically effective amount of the compounds and one ormore pharmaceutically acceptable carriers, excipients, or diluents(i.e., agents), which are nontoxic to the cell or mammal being exposedthereto at the dosages and concentrations employed. Often aphysiologically acceptable agent is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; proteins, such as serum albumin, or gelatin; hydrophilicpolymers such as polyvinylpyrrolidone; amino acids such as glycine,glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates Including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The disclosed pharmaceutical compositions may be immunogeniccompositions that optionally include adjuvants. For example, thecompositions may include oxCaMKII or a immunogenic fragment thereof andoptionally may include an adjuvant. The term “adjuvant” refers to acompound or mixture that enhances the immune response to an antigen. Anadjuvant can serve as a tissue depot that slowly releases the antigenand also as a lymphoid system, activator that non-specifically enhancesthe immune response. For example, adjuvants may include vitamin Eacetate solubilisate, aluminum hydroxide, aluminum phosphate or aluminumoxide, (mineral) oil emulsions, non-ionic detergents, squalene andsaponins. Other adjuvants which may be used include an oil basedadjuvants such as Freund's complete adjuvant (FCA), and Freund'sincomplete adjuvant (FLA). Other adjuvants include olefin cross-linkedunsaturated carboxylic acid polymers, such as cross-linked acrylic acidpolymers. As used herein the term “cross-linked acrylic acid polymer”refers to polymer and copolymers formed from a monomer mixture whichincludes acrylic acid as the predominant monomer in the mixture.Examples of suitable cross-linked acrylic acid polymers include thosecommercially available under the tradenames Carbopol® 934P and Carbopol®971 (available from B.F.Goodrich Co., Cleveland, Ohio).

The presently disclosed antibodies and antigen binding fragments thereofmay be utilized in methods for monitoring the course of a diseaseassociated with elevated levels of oxCaMKII in a subject. The methodsmay include evaluating the level of oxCaMKII in a series of biologicalsamples obtained at different time points from a subject, where a changein the level of oxCaMKII over time may be utilized to characterize adisease in the subject. For example, an increase in the level ofoxCaMKII over time may be indicative of progression of the disease, anda decrease in the level of oxCaMKII over time may indicate a regressionof the disease.

The presently disclosed antibodies and antigen binding fragments thereofalso may be utilized in methods for monitoring a therapeutic treatmentof a disease associated with elevated levels of oxCaMKII. The methodsmay include evaluating the level of oxCaMKII in a series of biologicalsamples obtained at different time points from a subject undergoing atherapeutic treatment for a disease associated with elevated levels ofoxCaMKII, where a change in the level of oxCaMKII over time may beutilized to characterize aspects of the therapeutic treatment includingefficacy and undesirable side effects. For example, a decrease in thelevel of oxCaMKII over time may indicate an effective therapeuticoutcome. Alternative, an increase in the level of oxCaMKII over time mayindicate negative or undesirable side effects associated with thetherapy. For example, the disclosed methods are utilized to detectoxCaMKII in a biological sample from a patient where the patient has oris at risk for developing a disease or condition, selected from a groupconsisting of a cardiac disease, cancer, premature aging,atherosclerosis, Alzheimer's disease, or sepsis. The method further mayinclude: (c) administering a therapeutic agent to the patient (or notadministering a therapeutic agent to the patient) based on detectingoxCaMKII (or not detecting oxCaMKII) in the biological sample from thepatient. With respect to the role of CaMKII in cardiac arrhythmia,reference is made to Erickson et al., “CaMKII and its role in cardiacarrhythmia,” J. Cardiovas. Electrophysiol. 2008 December; 19(1:2):1332-6, Epub 2008 September, the content of which is incorporated hereinin its entirety.

ILLUSTRATIVE EMBODIMENTS

The following embodiments are illustrative and are not intended to limitthe scope of the disclosed subject matter.

Embodiment 1

A purified antibody or antigen-binding fragment thereof that bindsspecifically to oxidized calcium/calmodulin-dependent protein kinase II(oxCaMKII).

Embodiment 2

The antibody or antigen-binding fragment thereof according to embodiment1, wherein the antibody is a human, mouse, rat, guinea pig, rabbit, dog,cat, pig, goat, horse or cow antibody.

Embodiment 3

The antibody or antigen-binding fragment thereof according to embodiment1, wherein the antibody is monoclonal.

Embodiment 4

The antibody or antigen-binding fragment thereof according to embodiment1, wherein the antibody is polyclonal.

Embodiment 5

The antibody or antigen-binding fragment thereof according to embodiment1, wherein the antibody is chimeric.

Embodiment 6

The antibody or antigen-binding fragment thereof according to embodiment1, wherein the antibody is humanized.

Embodiment 7

The antibody or antigen-binding fragment thereof according to embodiment1, wherein the antibody is a human antibody.

Embodiment 8

The antibody or antigen-binding fragment thereof according to embodiment1, wherein the antigen binding fragment is an Fab fragment.

Embodiment 9

The antibody or antigen-binding fragment thereof according to embodiment1, wherein the antigen binding fragment is an F(ab′)₂ fragment.

Embodiment 10

The antibody or antigen-binding fragment thereof according to embodiment1, wherein the antigen binding fragment is a dAb fragment.

Embodiment 11

The antibody or antigen-binding fragment thereof according to embodiment1, wherein the antigen binding fragment is a single chain Fv.

Embodiment 12

The antibody or antigen-binding fragment thereof according to embodiment1, wherein the antibody or antigen-binding fragment binds specificallyto a peptide consisting of an amino acid sequence selected from theconsensus amino acid sequence {S,C} {H,Q}RSTVAS {C,M}MHRQETV{D,E} (SEQID NO:4) in which the cysteine or methionine at position 9 is oxidizedand the methionine at position 10 is oxidized.

Embodiment 13

The antibody or antigen-binding fragment thereof according to embodiment1, wherein the antibody or antigen-binding fragment binds specificallyto oxCaMKII delta having oxidized methionine residues at positions 281and 282.

Embodiment 14

A labeled antibody or antigen-binding fragment thereof comprising theantibody or antigen-binding fragment thereof according to embodiment 1and a label.

Embodiment 15

An isolated CaMKII polypeptide or an immunogenic fragment thereof,comprising oxidized methionine residues at amino acid positions 281 and282.

Embodiment 16

A method of preparing antisera that binds specifically to oxCaMKII, themethod comprising: (a) administering to an animal a compositioncomprising CaMKII or an immunogenic fragment thereof having one or moreoxidized amino acids selected from a group consisting of oxidizedmethionine and oxidized cysteine; (b) and isolating antisera from theanimal.

Embodiment 17

The method of embodiment 16, wherein the composition further comprisesan adjuvant.

Embodiment 18

The method of embodiment 16, wherein the CaMKII or an immunogenicfragment thereof is prepared by a process comprising treating the CaMKIIor an immunogenic fragment thereof with an oxidizing agent.

Embodiment 19

An isolated cell that produces antibody that binds specifically tooxCaMKII.

Embodiment 20

The isolated cell of embodiment 19, wherein the cell is a hybridoma or aplasmacytoma.

Embodiment 21

A method of detecting oxCaMKII, comprising: (a) contacting a biologicalsample from a patient with an antibody or antigen-binding fragmentthereof that binds specifically to oxCaMKII and forms a complex; and (b)detecting the complex.

Embodiment 22

The method of embodiment 21, wherein the biological sample is blood orplasma.

Embodiment 23

The method of embodiment 21, further comprising: (c) characterizingheart disease in the patient.

Embodiment 24

The method of embodiment 21, wherein the patient has or is at risk fordeveloping a disease or condition selected from a group consisting of acardiac disease or condition, cancer; premature aging, atherosclerosis,Alzheimer's disease, or sepsis, and the method further comprises: (c)administering a therapeutic agent based on detecting or not detectingoxCaMKII.

Embodiment 25

The method of embodiment 21, wherein the patient is undergoing therapywith an angiotensin converting enzyme (ACE) inhibitor and the methodfurther comprises: (c) modulating the therapy based on detecting or notdetecting oxCaMKII.

Embodiment 26

The method of embodiment 25, wherein modulating the therapy comprisesincreasing dosage of the ACE inhibitor.

Embodiment 27

The method of embodiment 25, wherein modulating the therapy comprisesdecreasing dosage of the ACE inhibitor or ceasing the therapy.

Embodiment 28

The method of embodiment 21, wherein the patient is undergoinganti-cancer therapy and the method further comprises: (c) modulating thetherapy based on detecting or not detecting oxCaMKII.

Embodiment 29

The method of embodiment 28, wherein the anti-cancer therapy is selectedfrom a group consisting of radiation therapy, chemotherapy, nutritionaltherapy, and combinations thereof.

Embodiment 30

A kit for detecting oxCaMKII, comprising: (a) an antibody orantigen-binding fragment thereof that binds specifically to oxCaMKII toform a complex; and (b) a label for detecting the complex.

Embodiment 31

A method of treating or preventing structural heart disease in a patientcomprising administering to the patient an antibody or antigen-bindingfragment thereof that binds specifically to oxCaMKII.

Embodiment 32

The method of embodiment 31, wherein the antibody or antigen-bindingfragment binds specifically to oxCaMKII delta having oxidized methionineresidues at positions 281 and 282.

Embodiment 33

A pharmaceutical composition comprising: (a) an antibody orantigen-binding fragment thereof that binds specifically to oxCaMKII;and (b) a pharmaceutically acceptable carrier.

Embodiment 34

A method of treating or preventing structural heart disease in a patientcomprising administering a therapeutic agent that specifically inhibitsoxidation of calcium/calmodulin-dependent protein kinase II.

Embodiment 35

A method for treating or preventing structural heart disease in apatient comprising increasing methionine sulfoxide reductase activity inthe patient.

EXAMPLES

The following Examples are illustrative and are not intended to limitthe disclosed subject matter. The experiments were performed using themethodology described below. With respect to the following Examples,reference is made to Erickson e.g., “A dynamic pathway forcalcium-independent activation of CaMKII by methionine oxidation,” Cell,2008 May 2; 133(3):397-9, the content of which is incorporated herein byreference in its entirety. Direct Oxidation Results in Ca²⁺ IndependentActivation of CaMKII,” to be published in Cell, projected publicationdate of May 2, 2008.

Example I Direct Oxidation Results in Ca²⁺ Independent Activation ofCaMKII A. Summary

Calcium/calmodulin (Ca²⁺/CaM)-dependent protein kinase II (CaMKII)couples increases in cellular Ca²⁺ to fundamental responses in excitablecells. CaMKII was identified over twenty years ago by activationdependence on Ca²⁺/CaM, but recent evidence shows CaMKII activity isalso enhanced by pro-oxidant conditions. The data presented heredemonstrates that oxidation of paired regulatory domain methionineresidues sustains CaMKII activity in the absence of Ca²⁺/CaM. CaMKII isactivated by angiotensin II (AngII) induced oxidation, leading toapoptosis in cardiomyocytes, both in vitro and in vivo. CaMKII oxidationis reversed by methionine sulfoxide reductase A (MsrA), and MsrA^(−/−)mice show exaggerated CaMKII oxidation and myocardial apoptosis,impaired cardiac function, and increased mortality alter myocardialinfarction. These data demonstrate a novel, dynamic mechanism for CaMKIIactivation by oxidation and highlight the critical importance ofoxidation-dependent CaMKII activation to AngII and ischemic cardiacapoptosis.

B. Introduction

The multifunctional calcium/calmodulin (Ca²⁺/CaM)-dependent proteinkinase II (CaMKII) couples increases in Ca²⁺ to activation of ionchannels (Grueter e.g., 2006), gene transcription (Backs e.g., 2006),and apoptosis (Zhu e.g., 2003; Yang e.g., 2006). CaMKII is activated byenhanced intracellular Ca²⁺ from beta-adrenergic receptor (PAR)stimulation (Zhang e.g., 2005). Excessive βAR stimulation causesapoptosis by a Ca²⁺, CaMKII, and caspase-3 dependent pathway (Zhu e.g.,2003). The CaMKII holoenzyme is assembled from subunits containing threekey domains, the association domain, which directs multimeric assembly,the regulatory domain, which controls enzyme activation andautoinhibition, and the catalytic domain, which performs the kinasefunction of CaMKII. Under resting conditions CaMKII is inactive, butupon binding Ca²⁺/CaM a conformational change relieves theautoinhibitory effect of the regulatory domain on the kinase domain,activating the enzyme (Hudmon and Schulman, 2002; Rosenberg e.g., 2005).In the sustained presence of Ca²⁺/CaM, CaMKII undergoes intersubunitautophosphorylation at T287 (or 286; specific numbering is isoformdependent), resulting in Ca²⁺/CaM independent activity (Hudmon andSchulman, 2002). T287 lies within the autoinhibitory region of CaMKII,and autophosphorylation at T287 produces Ca²⁺ autonomous activity bypreventing re-association of the kinase domain by the autoinhibitoryregion (Hudmon and Schulman, 2002). Interconversion betweenCa²⁺-dependent and Ca²⁺-independent forms is a critical property ofCaMKII that allows transformation of a transient Ca²⁺ stimulus intosustained physiological or disease-causing activity.

CaMKII activity may also increase in pro-oxidant cellular environments(Howe e.g., 2004; Zhu e.g., 2007), suggesting CaMKII has broaderfunctionality than originally envisioned by connecting ‘upstream’oxidant stress and Ca²⁺ signals to ‘downstream’ cellular responses.Based upon the previously recognized structure-activity response ofCaMKII to T287 phosphorylation, it was hypothesized that oxidationdirectly modifies the autoinhibitory motif to confer Ca²⁺/CaMindependent CaMKII activity by a mechanism analogous toautophosphorylation. A novel direct molecular mechanism for reactiveoxygen species (ROS)-dependent, Ca²⁺ independent CaMKII activation bymodification of M281/282 was identified. These findings show that directactivation of CaMKII by ROS engenders Ca²⁺ autonomous activity, a clearbut previously unrecognized molecular mechanism by which CaMKII canintegrate Ca²⁺ and ROS signals.

Elevated levels of ROS have been measured and contribute to adverseoutcomes after myocardial infarction (Kinugawa e.g., 2000) and in modelsof heart failure (Maack e.g., 2003). Angiotensin II (AngII) alsoincreases ROS in heart (Doerries e.g., 2007), while AngII antagonistdrugs are a mainstay for reducing mortality in patients with structuralheart disease (Pfeffer e.g., 1992; Pfeffer e.g., 2003). It washypothesized that CaMKII is a downstream signal for ischemic and AngIIstimulated apoptosis in heart and that CaMKII responses were dependentupon this newly identified M281/282 activation mechanism. Methioninesulfoxide reductase A (MsrA) specifically reverses Met oxidation,suggesting that MsrA^(−/−) mice would show increased CaMKII oxidationafter AngII and ischemic stress. The data presented here demonstratethat CaMKII inhibition protects against AngII initiated apoptosis inheart and that pathological AngII responses recruit CaMKII activity byM281/282 oxidation in vitro and in vivo. MsrA^(−/−) mice show increasedCaMKII oxidation and apoptosis with AngII and ischemia and increasedmortality, greater left ventricular dilation and worse in vivomechanical function after myocardial infarction, compared to controls.The data also establish CaMKII as a downstream signal for AngII andischemic stress and establish ROS modification of CaMKII at M281/282 asa dynamic mechanism for regulating myocardial responses to common formsof heart disease.

C. Methods

1. Mouse Models

Mice lacking the p47 gene (p47^(−/−)) were purchased from Jackson Labs.Mice lacking the MsrA. (MsrA^(−/−)) were supplied by NIH (Bethesda,Md.). Mice with genetic CaMKII inhibition (AC3-I) were generated by usas previously described (Zhang e.g., 2005).

2. CaMKII Activity Assays and Protein Analysis

Mutant CaMKII cDNAs were generated using a QuikChange site-directedmutagenesis kit (Stratagene). CaMKIIδ (GenBank #NP_(—)001020609) wasgenerated using the Bac-to-Bac baculovirus system (Invitrogen) andpurified on a calmodulin-agarose column. For CaMKII activity assays,purified CaMKII was pretreated with 200 μM CaCl₂ and 1 μM CaM on ice for1 minute. The protein was then exposed to ATP, H₂O₂, or water at thedescribed concentrations for 10 minutes. Samples exposed to ATP or H₂O₂were then treated with 10 mM EGTA for 10 minutes. CaMKII activity wasmeasured as a function of ³²P-ATP incorporation into a syntheticsubstrate (syntide-2) at 30° C., as previously described (Wu e.g.,2002).

3. Oxidized M281/282 Immune Serum Production and Immune Staining

Antigenic peptide with the sequence CQRSTVASMMHRQETVD (SEQ ID NO:5)(Epitomics, Inc.) was generated and exposed to 100 μM H₂O₂ for one hour.Rabbits were immunized and antiserum titer was monitored using standardELISA procedures. Antiserum was collected after 75 and 96 days.Commercial antibodies were used for blots and immunostaining for total(Stressgen Biotechnologies) and phosphorylated (Santa Cruz) CaMKII.

4. Detection of ROS

Changes in ROS levels in cultured primary cardiac myocytes after agoniststimulation were measured using the fluorogenic probe dihydroethidium(DHE, 5 μM, Molecular Probes), as previously described (Zimmerman e.g.,2004). DHE fluorescent images were acquired using confocal microscopy(Zeiss LSM510).

5. Intracellular Calcium Concentration Measurements

Intracellular calcium concentration was assessed by Fura-2 fluorescenceratio imaging using a microscopic digital imaging system (PhotonTechnology International), as described previously (Sharma e.g., 1995).Briefly, cultured primary cardiac myocytes were loaded with theCa²⁺-specific dye Fura-2 by incubating with 1 μM Fura-2AM (MolecularProbes) at 37° C. for 30 minutes. [Ca²⁺]_(t) values over the entire cellwere calculated from the 340/380-nm ratio images of Fura-2 fluorescencecaptured before, during and after Iso (100 nM) or AngII (100 nM)stimulation.

6. Fluorescence Measurements

Spectra were collected at 30° C. using a Fluorolog 3 (Jobin Yvon,Horiba) spectrofluorometer. For intrinsic fluorescence shiftexperiments, excitation wavelength was 270 nm. Emission spectra weregenerated at Iran increments from 280 nm to 400 nm. Background traceswere subtracted from CaMKII spectra to eliminate the contribution fromintrinsic fluorescence of CaM. For fluorescence anisotropy experiments,baseline traces of 100 nM dansylated CaM in 15 mM HEPES buffer, pH 7.2were measured at baseline and after the addition of 200 μM CaCl₂ at 60s. At 180 s, 100 nM purified CaMKII was added to the CaM solution. Forsome trials, CaMKII became phosphorylated by the addition of 10 mM ATP.100 μM H₂O₂ or an equivalent volume of buffer was added at 250 s.Finally, addition of 10 mM EGTA at 300 s was used to remove free calciumfrom the solution, uncoupling CaM/CaMKII binding.

7. Cardiomyocyte TUNEL Immunostaining

Myocyte isolations from neonatal mouse or rat pups were modified frompreviously described methods (Mohler e.g., 2007). To generate theprimary cardiomyocyte cultures, hearts were dissected from P1 animalsand placed in 1 mL of Ham's F-10. Atrial tissue was removed and theventricular chambers were rinsed to remove any remaining blood. Heartswere transferred into 1.5 mL of 0.05% Trypsin, 200 μM EDTA in Ham's F-10medium (Mediatech). Hearts were minced into approximately 20 smallpieces using forceps and small scissors and incubated in theTrypsin/EDTA medium at 37° C. Following 15 min, the heart pieces weremixed by a Pasteur pipette and incubated for an additional 15 min. Amixture of 200 μL of soybean trypsin inhibitor (2 mg/mL; Worthington)and 200 μL of collagenase (0.2 mg/mL; 1980 units/mg; Sigma) wasincubated with the cells for 35-50 min at 37° C. The cell suspension waspelleted, resuspended in “Complete Medium” (40% DMEM, 40% Ham's F-10,20% FCS), and plated on plastic dishes. Following five hours, thenon-adhered cells (cardiomyocytes) were aspirated from the plate,pelleted, resuspended in Complete Medium, and plated on Mattek tissueculture plates (fibronectin-coated; Roche). Cardiomyocytes were washedwith Ham's F-10 and Medium was replaced with “Defined Medium” to preventthe growth of fibroblasts. 100× Defined Medium consists of 100 μg/mLinsulin, 500 μg/mL transferrin, 100 nM LiCl, 100 nM NaSeO₄, and 10 nMthyroxine. To ensure that pure populations of cardiomyocytes wereobtained, cultures were immunolabeled with alpha-actinin Ig(cardiomyocyte-specific marker). Only cultures with >90% cardiomyocyteswere used in experiments. (See FIG. 12.) Lentiviral treatments (shRNA,rescue constructs) and apoptosis inducing agents (Iso, AngII) were usedfor 24 hours. Cells were fixed in 4% paraformaldehyde, permeabilized in0.1% Triton X-100 and sodium citrate, and stained using In Situ celldeath detection kits, TMR Red (Roche). TUNEL stain assays wereinterpreted as previously described (Yang e.g., 2006). Nuclei wereco-stained with DAPI. Investigators were blinded to the genetic identityand treatment of the mice in all studies.

The CaMKIIδ-specific shRNA target sequence was selected based on thealgorithm of Chalk, Wahlestedt, and Sonnhammer and others (Hammond e.g.,2000; Bernstein e.g., 2001; Brummelkamp e.g., 2002). Five targetsequences were tested and the most effective target shRNA was used forexperiments in this manuscript (CaMKIIδ 920-938 CUAUGCUGGCUACGAGAAA (SEQID NO:6)). Sense and anti-sense sequences were created and ligated intoa modified pFIV lentiviral vector (SBI). For rescue experiments,full-length rat CaMKIIδ was cloned into pCDH1-MCS1-EF1-copOFP (SBI) andcompletely sequenced. The resulting construct was then modified to beshRNA resistant by engineering conserved residue wobble base changes atfour positions in the template (CTG[A]-GCT[C]-ACG[A]-AGA[G] (SEQ IDNO:7))→LATR to LATR. The resulting construct was then fullyre-sequenced. The fidelity of the viral knock-down and rescue constructwere confirmed in primary cardiomyocytes.

9. Virus Generation

The most efficacious shRNA construct was engineered into the pFIVlentiviral vector and packaged into viral pseudoparticles using theSystem Biosciences viral packaging kit (SBI LV100A-1). Specifically theconstructs were co-transfected with packaging plasmid mix from the kitinto HEK293 FT cells using Effectene anionic lipid transfection reagent.After 16 hours the serum free media was changed to fresh DMEM with 10%FBS and 1% penicillin/streptomycin. 48 hours later the pseudoparticlecontaining supernatant was concentrated using Amicon Centriplus YM-30columns spun at 2500×g for 3 hours at 4° C. The concentrate was thendispensed into 200 μl aliquots and stored at −80° C. Experiments weredone with cells cultured 48 hours after infection.

10. Heart Section TUNEL Immunostaining

Mice were anesthetized with ketamine (87.5 mg/kg) and xylazine (12.5mg/kg), and a small incision was made in the skin near the spine,approximately 1 cm above the hip. Minipumps containing saline or AngII(3 mg/kg/day) were inserted and the skin was sutured closed. Other micewere given, daily injections of Iso (30 mg/kg/day, intraperitoneal) for7 days. Animals were then sacrificed and the heart was removed, fixed,and embedded in paraffin. Transverse heart sections of 5 μm thicknesswere made. To confirm the identity of cardiomyocytes, heart sectionswere immunolabeled with alpha-actinin Ig (cardiomyocyte-specificmarker). Only sections with >90% cardiomyocytes were used inexperiments. (See FIG. 13.) Paraffin was removed, and TUNEL staining wasperformed using In Situ cell death detection kits, TMR Red (Roche).Nuclei were co-stained with DAPI. To determine percent of TUNEL positivenuclei, whole heart sections were divided into four quadrants and oneimage was taken at random within each quadrant. In the case of sectionsfrom post-MI mice, an average of 50% of the heart quadrants were fromportions of the heart adjacent to the MI. An investigator blinded to theidentity and treatment of the mice counted the number of total and TUNELpositive cells in each image.

Other sections were treated with either a general CaMKII antibody oroxidized CaMKII antiserum. Nuclei were co-stained with DAPI. Identity ofcardiomyocytes was confirmed by co-stain with an antibody againstα-actinin. Images were quantified for relative staining intensity usingImage J (NIH, USA). Each image was divided into four quadrants, and onerectangular area of equal size and shape was randomly placed within thecytosolic area of each quadrant, excluding DAPI-stained nuclei. Relativeintensity of an image was judged to be the mean of the intensitymeasurements taken in each quadrant. Both the investigators andtechnical personnel assigned to immunostaining and quantification wereblinded to the genetic identity and treatment of the mice in allstudies.

11. Myocardial Infarction and Echocardiogram

Mice were anesthetized with ketamine/xylazine (87.5/12.5 mg/kg,respectively). Mice were then intubated and ventilated with room air(150 μl tidal volume, 120 breaths/min). An incision was made in the3^(rd) intercostal space, and the ribs were retracted to expose theheart. The pericardial sac was opened, and the left anterior descending(LAB) branch of the coronary artery was ligated using 8-0 ethilon suture(Ethicon) along the anterolateral border of the heart as close to theleft atrial appendage as possible. Successful ligation of the artery isconfirmed by blanching of the myocardium. A chest tube was inserted intothe thorax, and the muscle and skin were sutured closed around the tubeto form a seal. Negative pressure was applied to reduce thepneumothorax. Mice were then extubated.

Transthoracic echocardiograms were recorded in conscious sedated mice asdescribed previously (Weiss e.g., 2006), using a 15 MHz probe connectedto a Sonos 5500 imager (Phillips Medical Systems, Bothell, Wash.) at aframe rate of 180-200 frames per second. Images were acquired by anoperator blinded to mouse genotype and were analyzed offline usingcustom-designed software (Ereeland Medical Systems, Louisville, Colo.).

12. Cysteine Protection and Ellman's Reagent Assays

CaMKII was pretreated with 10 mM iodoacetic acid to block oxidation ofavailable cysteine residues. Activity was determined as described inmain text. To test the efficacy of cysteine residue blockage, acolorometric assay was performed with or without iodoacetic acid.Samples of CaMKII were incubated with Ellman's reagent (benzoic acid,Pierce) for 15 minutes, and the absorbance was measured at 412 nm in aspectrophotometer. Molar ratios of available cysteine residues weredetermined by comparing to a standard curve of purified cysteinesamples. Control, experiments were performed with a peptide known tocontain exactly one cysteine.

13. LC-MS-MS Analysis and Protein Identification

The synthetic peptide CQRSTVASMMHRQETVD (SEQ ID NO:5), with and withoutH₂O₂ treatment, was subjected to in solution trypsin digestion. LC-MS-MSanalysis of the resulting peptides was performed using a Thermo FinniganLTQ ion trap mass spectrometer as previously described (Grueter e.g.,2006) except that the mass spectrometer was equipped with a ThermoMicroAS autosampler and the peptides were separated on a packedcapillary tip with CIS resin (Jupiter C₁₈, 5 micron, 300 angstrom,Phenomonex. Torrance, Calif.) using an inline solid phase extractioncolumn that was 100 μm×4 cm packed with the same C18 resin using a fritgenerated with liquid silicate Kasil 1. The mass spectrometer was tunedprior to analysis using the synthetic peptide TpepK. (AVAGKAGAR (SEQ IDNO:8)). Typical tune parameters were as follows: spray voltage ofbetween 1.8 KV, a capillary temperature of 150° C., a capillary voltageof 50V and tube lens 100V. One full MS scan from 400-2000 m/z wasacquired followed by the acquisition of MS/MS scans in a targetedfashion collecting MS/MS spectra for the doubly charged version of theSTVASMMHR peptide (SEQ ID NO: 1) (510.24 m/z) and it's correspondingoxidized species (518.24, 526.24, 534.24, 542,24 m/z). Fragment iontraces were extracted for each m/z value utilizing the y₅ and y₆ ionsfor relative quantification experiments. MS/MS spectra were collectedusing an isolation width of 3 m/z, an activation time of 30 ms, andactivation Q of 0.250 and 30% normalized collision energy using 1microscan and maximum injection time of 100 for each scan. For therelative quantification experiments, targeted. MS/MS analyses of threenormalization peptides, LLKHPNIVR (SEQ ID NO:9), GAFSVVR (SEQ ID NO:10)and IPTGQEYAAK (SEQ ID NO:11) (363.89, 368.21 and 539.28 m/z,respectively) in the same run as the methionine containing peptides wereperformed. Fragment ion traces for the normalization peptides wereextracted utilizing the four most abundant ions (two most abundant ionsfor GAFSVVR) in each spectrum that were either b- or y-ions. Thepeptides were quantified by extracting the fragment Ion traces,integrating the peaks in Xcalibur to obtain peak area for both themethionine containing peptides and the normalization peptides. Once thepeak areas were extracted, the methionine containing peptides werenormalized by dividing the peak area by each of the normalizationpeptides and then taking the ratio of each of these normalized signalsin the treated vs. non-treated samples. The ratios for each of thenormalized signals were then averaged for each replicate analysis.

14. Caspase-3 Activity Assays

Cells were lysed in assay lysis buffer (50 mM Tris-HCl pH 7.5, 100 mMKCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 0.5 mM benzamidine, 20mg/L leupeptin, 1 μM microcystin, 20 mM sodium pyrophosphate, 50 mM NaF,and 50 nM sodium β-glycerophosphate) and total protein content wasdetermined by Biuret assay. Caspase-3 activity was determined byEnz-Chek Caspase-3 kit (Invitrogen).

15. Statistical Analysis

Statistical significance for mortality study was determined bychi-squares test. All other statistical significance was determined byOne-Way ANOVA with post hoc Bonferonni tests. A p value of <0.05 wasconsidered statistically significant. All results are presented asmean±SEM.

D. Results

1. Oxidation Directly Activates CaMKII

CaMKII is activated by Ca²⁺/CaM, but autophosphorylation at T287sustains catalytic activity after dissociation of Ca²⁺/CaM (FIG. 1A)because the negatively charged phosphate prevents reassociation of thecatalytic domain and autoinhibitory region (Hudmon and Schulman, 2002).CaMKII activity may also be enhanced by pro-oxidant conditions (Zhue.g., 2007); it therefore was hypothesized that oxidation of theregulatory domain in the vicinity of T287 could sustain CaMKII catalyticactivity by an analogous mechanism. Exposure of purified CaMKII to H₂O₂in the absence of any pre-treatment yielded no discernable CaMKIIactivity (FIG. 1B). However, exposure to H₂O₂ after pretreatment withCa⁺/CaM yielded persistent CaMKII activation even in the presence ofEGTA. These data suggest that Ca²⁺/CaM binding exposed a key segment ofCaMKII for oxidation, and that oxidation interfered with the interactionof the autoinhibitory and catalytic domains. Activation of wild type(WT) CaMKII by H₂O₂ was close-dependent (FIG. 1C). The concentration ofEGTA used was sufficient to block CaMKII activity without the additionof H₂O₂ (FIG. 1B), suggesting that activity observed in the pro-oxidantcondition was independent of sustained Ca²⁺/CaM binding.

Pretreatment with Ca²⁺/CaM was also necessary forautophosphorylation-dependent CaMKII activation, indicating thatautophosphorylation and oxidation of CaMKII occur by parallelmechanisms. CaMKII bearing a T287A substitution underwent normalCa²⁺/CaM-dependent activation but did not maintain persistentCa²⁺-independent activity in the presence of ATP (FIG. 1D). However, theT287A mutant was activated by H₂O₂ (FIG. 1C), and the extent of thisactivation was statistically indistinguishable at ail but the highestconcentration of H₂O₂ tested (1 μM). These observations are interpretedas evidence that activation of CaMKII by ROS and autophosphorylationoccur by a similar mechanism, but by independent modifications to nearbysites. Activation of the kinase by either mechanism requires the enzymeto be initially ‘opened’ by Ca²⁺/CaM to allow access to theautoinhibitory domain for oxidation or autophosphorylation (FIG. 1A, E).Either of these modifications can prevent subsequent interaction of theautoinhibitory region with the catalytic domain, providing for sustainedCa²⁺-independent activation of CaMKII. Consistent with these ideas,direct, measurements of intrinsic fluorescence revealed thatautophosphorylation and oxidation of CaMKII independently induce similarconformational changes in CaMKII. (See FIG. 2).

2. Chromatographic Analysis of CaMKII and M281/282V Mutant AfterTreatment with ATP or H₂O₂

Proteomic analysis of the synthetic peptide that contains the 281/282methionine residues was used to probe for oxidative modification upontreatment with H₂O₂. Because an internal standard was not available, thechange in oxidation with the synthetic peptide was not quantified. Tocompensate, the peptides for MS/MS fragmentation were targetedthroughout the ran. The number of spectra observed for a peptide orprotein previously has been shown to be correlate with peptide andprotein levels (Liu e.g., 2004). By targeting the peptides, multiplespectra across each peak were observed. Based on the chromatographictraces and on the change in the number of observed spectra, a cleardecrease in the unoxidized form coupled with an increase in the variousoxidized forms of this peptide was observed. (Data not shown). The MS/MSfragmentation spectra were verified by manual inspection of the SEQUESTresults.

In addition to the synthetic peptide, the peptide containing the 281/282methionine residues was analyzed after treatment of the whole proteinwith H₂O₂ followed by trypsin cleavage. To better quantify the change inthe oxidation, several other unmodified tryptic peptides produced fromthe digestion (LLKHPNIVR (SEQ ID NO:9), GAFSVVR (SEQ ID NO:10) andIPTGQEYAAK (SEQ ID NO:11)) were assessed to normalize the signal The m/zvalues then were targeted throughout the chromatogram; fragment iontraces for a pseudo-MRM trace were extracted; and the peak area of theoxidized peptide to the normalizing peptides were compared to normalizethe signal. By taking the ratio of the normalized signals, the relativechange in oxidation of this peptide upon hydrogen peroxide treatment wasdetermined. (See Tables 1 & 2).

TABLE 1 Oxidation of synthetic peptide methionines uponH₂O₂ treatment and trypsin digestion. Spectral counts^(a) PeptideUntreated H₂O₂ treated STVASMMHR 41 3 STVASMMHR + 1 Oxygen 13 0STVASMMHR + 2 Oxygen 0 3 STVASMMHR + 3 Oxygen 0 52 STVASMMHR + 4 Oxygen0 6 ^(a)Spectral counts are the number of times the spectrum for thecorresponding peptide was observed after filtration criteria wereapplied as described in Methods.

TABLE 2 Oxidation of tryptically digested methionine-containing peptides after H₂O₂ treatment of intact protein. PeptideRelative Change^(a) STVASMMHR 0.36 ± 0.06 STVASMMHR + 1 Oxygen 6.4 ± 1.9STVASMMHR + 2 Oxygen 115 ± 25  STVASMMHR + 3 Oxygen NQ^(b) STVASMMHR +4 Oxygen NO^(c) ^(a)Relative change was calculated as described in themethods and is reported as average ± standard deviation from threereplicate analyses. ^(b)Observed in H₂O₂ treated sample, absent incontrol sample, so not quantified ^(c)Not observed.

The MS/MS spectra of the oxidized forms of the peptide were identical tothose from the synthetic peptide, verifying that the oxidized peptidewas correctly identified. In addition to an increase in both singly anddoubly oxidized forms of this peptide, a triply oxidized peptide in theH₂O₂-treated protein that was absent in the untreated protein MS/MSspectrum was observed. Because there was an absence of this peptide inthe untreated sample, the relative increase in the triply oxidizedpeptide was not calculated.

3. Mutant Forms of CaMKII that are Resistant to Activation by OxidativeTreatment

Given these observations and the recognized susceptibility of methionineresidues to oxidation (Hoshi and Heinemann, 2001), mutants havingmethionine to valine substitutions for the paired residues (M281/282V)and for another methionine (M308V) in the CaM-binding region werecreated. These mutants were exposed to H₂O₂ and assayed for activity inthe presence of EGTA (FIG. 1C). The H₂O₂-dependent activation of CaMKIIwas preserved in the M308V mutant. However, oxidation-dependent CaMKIIactivity was completely abolished in the M281/282V and M281/282/308Vmutants. These data, obtained in cell free assay conditions, point todirect oxidation of the M281/282 pair as the primary H₂O₂-dependentactivation pathway for CaMKII. Importantly, all the methionine to valinemutants showed a normal activity response to autophosphorylation (FIG.1D), further supporting the concept that Ca²⁺ autonomous CaMKIIactivation by ROS or T287 autophosphorylation are independent events.While the paired methionine motif is conserved in the β, γ, and δisoforms of CaMKII, the neuronal α isoform substitutes a cysteineresidue for the first methionine of the pair (position 280 in CaMKIIα).The side chain of cysteine is also susceptible to oxidation. A M281Cmutant of CaMKIIδ was generated to mimic the substitution in CaMKIIα.Additionally, CaMKIIα was generated and purified. Both the M281C CaMKIIδmutant and the purified CaMKIIα were activated by H₂O₂, indicating thatthe cysteine substitution seen in CaMKIIα also supports ROS-dependentactivation (FIG. 1C). To further elucidate the role of M281 and M282 inROS-dependent activation, these sites were individually mutated. (SeeFIG. 3.) The M282V mutation completely ablated oxidation-dependentactivation, while the M281V mutation partially reduced activation by65%, indicating that a single oxidation event within the regulatorydomain is insufficient to activate CaMKII.

Autophosphorylation at T287 dramatically increases the binding affinityof CaMKII for CaM, a phenomenon known as “CaM trapping” (Meyer e.g.,1992). In the absence of ATP the Ca²⁺/CaM/CaMKII complex was veryrapidly dissociated following addition of EGTA, independent of the redoxstate, as measured by fluorescence anisotropy of dansylated CaM. (SeeFIG. 4A.) CaMKII exposure to H₂O₂ for 10 mm induced Ca²⁺/CaM-independentactivity (as in FIG. 1B), but also failed to induce CaM trapping (notshown). These observations indicate that under normal experimentalconditions, oxidation, of CaMKII is not sufficient to induce CaMtrapping. Dissociation of Ca²⁺/CaM from autophosporylated CaMKII and CaMwas significantly slower than from nonphosphorylated enzyme, consistentwith CaM trapping. However, pretreatment with H₂O₂ prior to EGTA had nosignificant effect on the dissociation kinetics. Thus, oxidation of CaMor CaMKII does not prevent or enhance CaM trapping by autophosphorylatedCaMKII. CaM trapping is reduced by phosphorylation of T306/307 (Colbran1993), suggesting that oxidation of M308 might prevent CaM trapping by aparallel mechanism. A significant slowing of dissociation of theCaM/CaMKII complex after H₂O₂ treatment of the M308 mutant was observed.(See FIGS. 4B, C.) These data suggest that the absence of CaM trappingduring oxidation is partly due to M308.

It seemed possible that conditions capable of oxidizing methionineresidues would also oxidize unprotected cysteine residues. Althoughmutation of methionine residues at 281 and 282 was sufficient tocompletely ablate ROS-dependent activation of CaMKII, a C290V mutant wascreated to determine whether this cysteine residue within the CaMKIIregulatory domain could also play a role. Both the Ca²⁺/CaM-dependentand ROS-dependent activity of the C290V mutant were indistinguishablefrom WT CaMKII. (See FIG. 4D.) This finding that oxidation of pairedamino acids (M281/282 in CaMKIIδ) were required for activation by H₂O₂support a view that oxidation of a lone residue is insufficient toconfer Ca²⁺/CaM autonomous CaMKII activity. In order to comprehensivelytest the potential role of all accessible cysteines in contributing tooxidation-dependent CaMKII activity, CaMKII activity responses to H₂O₂in the presence of iodoacetic acid were measured. Iodoacetic acid is areagent that blocks oxidation of unprotected cysteine residues (Zangerlee.g., 1992). Cysteine protected CaMKIIδ showed equivalent H₂O₂ activityresponses compared to CaMKIIδ without iodoacetic acid. (Data not shown.)An established colorometric assay was used to quantify the availablecysteine residues and verify that, cysteine protection by iodoaceticacid was effective. These results confirmed that most or all of the 11cysteines in CaMKIIδ were accessible to the Ellman's reagent afterCa²⁺/CaM binding, while treatment with iodoacetic acid blocked theaccessibility of cysteine residues to biochemical modification (data notshown). Taken together, these findings demonstrate that oxidativeactivation of CaMKIIδ is independent of cysteines.

4. Oxidation of CaMKII Occurs in vivo

A new immune serum against, oxidized M281/282 was prepared to detect ROSeffects on CaMKII in vivo. The fidelity of the antiserum was validatedusing purified CaMKII protein by immunoblotting against WT CaMKII andthe M281/282V mutant in control conditions and after treatment with H₂O₂or Ca²⁺/CaM/ATP. Blots were also assayed with a phospho- andsite-specific antibody against T287 (p-287). WT CaMKII exposed to H₂O₂after pretreatment with Ca²⁺/CaM showed significant reactivity tooxidized M281/282 antiserum, but untreated and T287-phosphorylatedCaMKII samples were not recognized by this antiserum. (See FIG. 5A). TheM281/282V mutant had minimal reactivity to this antiserum among thethree treatments. These findings demonstrated that phospho-T287 andoxidized M281/282 were immunologically distinct sites. Additionalimmunoblots were performed in which oxidized CaMKII was probed with theantiserum along with increasing concentrations of the peptide antigen(FIG. 5B). Band intensity decreased with increasing peptideconcentration, indicating that the immune serum was specific foroxidized CaMKII.

To determine the role of CaMKII oxidation In apoptosis, mice weretreated with saline, AngII, or isoproterenol (Iso) for one week, andtransverse heart, sections from these mice were probed for theproduction of oxidized CaMKII in vivo. WT mice treated with AngIIproduced more oxidized CaMKII than those treated with saline or Iso(FIG. 5C). Total CaMKII immunoreactivity remained constant regardless oftreatment. Conversely, mice lacking a critical subunit of NADPH oxidase(p47^(−/−)) did not show increased levels of oxidized CaMKII in responseto AngII. The p47^(−/−) mice do not assemble the ROS-producing complexNADPH oxidase (Munzel and Keaney, 2001), the main source of ROS due toAngII stimulation in many cell types (Lyle and Griendling, 2006). Heartsections from WT mice showed increased staining for T287-phosphorylatedCaMKII after AngII treatment, while p47^(−/−) mice were unaffected. (SeeFIG. 6). Other studies have suggested that protein phosphatase activityis decreased, by pro-oxidant conditions (Howe e.g., 2004), indicatingthe possibility of coordinate activation of CaMKII both by directoxidation at the Met281/282 sites and by phosphatase inactivationleading to increased phosphorylation at the T287 site.

Homogenized hearts from mice treated with saline, AngII, or Iso, andwhole heart lysates were analyzed by immunoblot for oxidized CaMKII.While total CaMKII was not significantly different among the threetreatment groups, heart lysates from mice treated with AngII showedsignificantly increased oxidized CaMKII levels (FIG. 5D). Takentogether, these findings demonstrate that oxidation of CaMKII occurs invivo, and that elevated levels of AngII increase CaMKII oxidation atM281/282 compared to saline or Iso.

5. AngII Triggers ROS Production and CaMKII-Dependent Apoptosis inCardiomyocytes

Given these results, it was hypothesized that cells deficient in ROSproduction or CaMKII activity would be resistant to AngII mediatedapoptosis. Cardiomyocytes from mice that express an inhibitory peptideagainst CaMKII (AC3-I, Zhang e.g., 2005) were treated with 100 nM AngIIfor 24 hours in parallel with isolated cardiomyocytes from WT andp47^(−/−) mice. AngII caused a significant increase in the percent ofTUNEL positive nuclei in WT cells but had no significant effect inp47^(−/−) or AC3-I cardiomyocytes (FIG. 6). Activity assays forcaspase-3, a downstream target enzyme in the CaMKII apoptotic signalingpathway in heart, recapitulated the results from the TUNEL assay (FIG.7A). Importantly, direct addition of ROS in the form of H₂O₂ causedsignificant apoptosis in p47^(−/−) cells, demonstrating that theirresistance to AngII-induced apoptosis is a result of impaired ability toproduce ROS rather than a lack of sensitivity to ROS. The apoptoticeffect of H₂O₂ in AC3-I cells was blunted by more than half compared toWT or p47^(−/−) cells (FIG. 7A), indicating the critical importance ofCaMKII activation to ROS and Iso-dependent apoptosis.

In order to validate the connection between AngII and ROS in thisexperimental model, isolated cardiomyocytes from WT and p47^(−/−) micewere treated with 100 nM AngII and monitored production of ROS byImaging DHE, a fluorescent reporter for superoxide and hydrogen peroxide(FIG. 7C). Wild-type cardiomyocytes were incubated with fura-2 AM, acell-permeant calcium indicator to observe changes in intracellular Ca²⁺([Ca²⁺]_(i)). Treatment with AngII caused a significant increase in ROSproduction in WT but not in p47^(−/−) cardiomyocytes (FIG. 7D). On theother hand, increases in [Ca²⁺]_(i) were significantly less after AngIIcompared to Iso treatment (FIG. 7E, F) for cardiomyocytes from both WTand p47^(−/−) mice. These data show AngII signaling predominantlyincreases ROS, while Iso predominantly increases [Ca²⁺]_(i) under theseexperimental conditions.

6. CaMKII Knockdown Prevents AngII- and Iso-Induced Apoptosis

In order to further test the role of CaMKII and specifically define theeffects of M281/282 on myocardial apoptosis, a knock down andreplacement strategy in cultured neonatal cardiomyocytes was utilized.Rat cardiomyocytes were cultured and treated with shRNA encodinglentivirus against rat CaMKIIδ. After 48 hours CaMKII expression wassignificantly reduced, as measured by immunoblot and activity assays(FIG. 8A). Cells were then transduced with lentivirus encodingshRNA-resistant WT or M281/282V mutant CaMKII. Control cells weretransduced with GFP encoding lentivirus. After 48 hours, cellstransduced with CaMKII rescue constructs showed significant recovery ofCaMKII expression compared to control cells. Addition of Ca²⁺/CaM tolysates from cells transduced with either CaMKII encoding virus hadsimilar total activity as native cells. However, H₂O₂-induced activitywas only rescued in cells expressing the WT CaMKII construct. Thesecellular studies support this earlier finding with molecular CaMKII(FIG. 1) by showing that oxidation of M281/282 is critical for ROStriggered CaMKII activity. In addition, this strategy createdcardiomyocytes expressing ROS-resistant CaMKII, providing a novel systemfor investigating ROS and CaMKII-dependent apoptosis.

Cardiomyocytes treated with shRNAs/CaMKIIδ encoding lentivirus wereexposed to saline, AngII or Iso as above (FIG. 8B). The apoptoticresponse to AngII and Iso was significantly attenuated in CaMKIIknockdown cells compared, to myocytes without shRNA. Moreover,expression of shRNA-resistant WT CaMKII fully rescued apoptoticresponses to both agonists (FIG. 8C). In contrast, expression of theROS-resistant M281/282V CaMKII mutant rescued the apoptotic response toIso but, importantly, failed to rescue the apoptotic response to AngIIafter 24 hours (FIG. 8C). Cells expressing the M281/282V CaMKII remainsusceptible to Iso-induced apoptosis (FIG. 8B, C), indicating thatelimination of these residues does not affect activation of CaMKII bycatecholamine stimulation. These cellular studies are performed in acomplex biological environment compared to studies with isolated CaMKII,but nevertheless support a concept that direct oxidation of CaMKII byAngII is sufficient to confer enhanced CaMKII activity and triggerapoptosis.

7. ROS Production and CaMKII Activity are Critical for AngII-MediatedCardiac Apoptosis in Vivo

Intracellular ROS levels increase dramatically in models of structuralheart disease (Hare, 2001), particularly those initiated by AngII (Tojoe.g., 2002). Stimulation by AngII leads to activation of the NADPHoxidase complex, increasing intracellular superoxide and hydrogenperoxide levels. To establish an in vivo context for these previousfindings and to test the role of CaMKII in AngII-stimulated cardiacapoptosis, p47^(−/−), AC3-I, and WT mice were treated with saline, AngIIor Iso for one week. Transverse heart sections from these mice werestained for evidence of apoptosis. After one week WT mice treated witheither AngII or Iso showed significant cardiac apoptosis, as determinedby TUNEL staining of heart sections (FIG. 9). The p47^(−/−) mice had nosignificant increase in cardiac apoptosis after treatment with AngII,most likely because these mice were unable to produce ROS in response toAngII stimulation (FIG. 7D). However, the p47^(−/−) mice showed apreserved apoptotic response to Iso, suggesting that Iso-inducedapoptosis occurs independently of oxidative stress generated by NADPHoxidase in vivo under these conditions. In contrast, the AC3-I mice withCaMKII inhibition were resistant to apoptosis induced by either AngII orIso, indicating that CaMKII is a necessary signal element for apoptosisinitiated by AngII or Iso in vivo.

8. Increased CaMKII Oxidation, Apoptosis, Cardiac Dysfunction and Deathin MsrA^(−/−) Mice

Methionine oxidation is specifically reversed by MsrA (Weissbach e.g.,2002), so it was hypothesized that MsrA^(−/−) mice would show enhancedvulnerability to AngII-mediated CaMKII oxidation and apoptosis. In orderto test this idea, MsrA^(−/−) and WT control mice were implanted withAngII or saline elating osmotic mini pumps. Hearts from MsrA^(−/−) micetreated with AngII in vivo showed significantly more CaMKII oxidation(FIG. 10A,B) and increased TUNEL staining (FIG. 10C) compared to salinetreated MsrA^(−/−) mice and to saline or AngII treated control hearts.The increased CaMKII oxidation by AngII in MsrA^(−/−) hearts showed thatCaMKII oxidation is dynamically regulated by MsrA in myocardium in vivo,and suggested that MsrA^(−/−) mice would be more vulnerable to severemyocardial stress due to increased methionine oxidation. Myocardialinfarction is the most common cause of sudden cardiac death and heartfailure in patients, and p47^(−/−) (Doerries e.g., 2007) and AC3-I mice(Zhang e.g., 2005) are protected from left ventricular dilation anddysfunction after myocardial infarction surgery, suggesting that ROSactivation of CaMKII may be important in myocardial infarction. In orderto test if CaMKII oxidation and apoptosis were regulated by NADPHoxidase and MsrA in the setting of myocardial infarction, MsrA^(−/−),p47^(−/−), and WT mice were subjected to myocardial infarction surgery.MsrA^(−/−) mice showed significantly more CaMKII oxidation (FIG. 11A,B)and myocardial apoptosis (FIG. 11C) compared to WT and p47^(−/−) mice.These data indicate CaMKII oxidation is dynamically regulated by NADPHoxidase and MsrA in the setting of myocardial infarction. Myocardialinfarction surgery was performed on a larger cohort of MsrA^(−/−) and WTcontrol mice to determine if increased CaMKII oxidation and apoptosis inMsrA^(−/−) mice translated into poorer functional outcomes. MsrA^(−/−)mice were significantly more likely to die after surgery compared to WTcontrols (FIG. 11D, p=0.0015) and exhibited significantly greater leftventricular dilation (FIG. 11E) and impaired systolic function (FIG.11F) compared to controls, demonstrating that methionine oxidationincreases the pathological impact of myocardial infarction in vivo.

E. Discussion

CaMKII was first identified by its dependence on Ca²⁺/CaM for activation(Schulman and Greengard, 1978). Later it was recognized, thatautophosphorylation at T287 modified the enzyme so that activitypersisted even in the absence of elevated Ca²⁺/CaM (Saitoh and Schwartz,1985; Lou e.g., 1986; Patron e.g., 1990). These findings unveil a newdimension to CaMKII signaling by showing that oxidation of M281/282 is adistinct molecular event, but with similar consequences to Thr287autophosphorylation for sustaining Ca²⁺/CaM independent activity.Oxidative activation likely is important in all known CaMKII isoforms,and relies upon paired, oxidation susceptible residues (MM in (β, γ andδ). Because of the ability of CaMKII to transition between Ca²⁺/CaMdependent and Ca²⁺/CaM independent species, CaMKII is considered a‘memory molecule’ for the history of Intracellular Ca²⁺ elevation. ROSfacilitation of Ca²⁺/CaM CaMKII activity suggests that the ability ofCaMKII to respond to Ca²⁺ elevation is enhanced in pro-oxidantconditions. Because increased CaMKII activity and oxidative stress areimplicated in a wide variety of physiological and disease processes,these findings have potentially broad implications for improvedunderstanding of connections between ROS and Ca²⁺ in multiple celltypes.

CaMKII is initially activated by Ca²⁺/CaM binding, which blocks theautoinhibitory association between the regulatory and catalytic domains.Phosphorylation of T287 blocks the reassociation of the regulatory andcatalytic domains, conferring Ca²⁺/CaM independent activity on theenzyme (Hudmon and Schulman, 2002). In this study, a novel mechanism forCaMKII activation by oxidation of M281/282 was discovered. As is thecase for T287-autophosphorylation, activating oxidation appears torequire that the regulatory domain is first exposed by Ca²⁺/CaM binding,whereupon oxidation at M281/282 leads to persistent Ca²⁺/CaM-autonomousactivation of CaMKII. Oxidation of methionine residues changes both thecharge and flexibility of their side chains (Hoshi and Heinemann, 2001),apparently leading to steric blockage of reassociation between theregulatory and catalytic domains. Surprisingly, cysteine residues, acommon target for oxidative regulation (Barford, 2004), do not appear toplay any role in oxidative activation of CaMKIIδ (or by inferenceCaMKIIβ or γ). The regulatory domain of all CaMKII isoforms contains asingle cysteine (C290 in CaMKIIδ), but oxidation of this unpairedcysteine is insufficient to activate CaMKIIδ. These data are alignedwith the concept that Ca²⁺/CaM-dependent, exposure of the regulatorydomain sets up the CaMKII molecule for subsequent modifications thatconfer persistent, Ca²⁺ independent activity.

These findings identified a previously unrecognized mechanism ofenhancing CaMKII by direct methionine oxidation, but oxidation mayaffect activity of kinases by multiple mechanisms. Pro-oxidantconditions can modify the activity levels of protein kinases by directand indirect mechanisms. For example, direct thiol oxidation within theATP binding pocket inhibits MEK kinase 1 activity (Cross and Templeton,2004). Oxidative stress can induce activation of ERK1/2 (Engers e.g.,2006), while oxidation-dependent inactivation of protein phosphatases(Tonks, 2006) and upstream kinase kinases, such as IKK-β (Reynaert e.g.,2006) can indirectly lead to increased kinase activity. The presentfindings that AngII increases both CaMKII oxidation andautophosphorylation suggest that ROS inhibition of phosphatases furtherenhances CaMKII activity responses to oxidant stress in vivo.

Autophosphorylation at T287 is reversed by phosphatase activity(Zhabotinsky, 2000; Hudmon and Schulman, 2002). Because phosphorylationis a readily reversible process, activation by autophosphorylationrepresents a tunable regulatory mechanism for CaMKII. Oxidation ofmethionine residues is also a reversible biochemical modification, andthe presence of methionine residues can confer functional sensitivity tooxidative stress (Santarelli e.g., 2006). Methionine sulfoxide reductase(Msr) reduces the side chain of methionine to its native state (Kryukove.g., 2002), and is therefore a critical defense mechanism againstcellular damage by oxidative stress. Mutant Drosophila overexpressingMsr had longer life spans, (Ruan e.g., 2002) while MsrA^(−/−) mice showincreased mortality in response to oxidant induced aging (Moskovitze.g., 2001). The importance of MsrA in various biological systemssuggests that reversible oxidation of methionine residues couldcomplement a Thr287 phosphorylation/dephosphorylation cycle by servingas a ROS responsive regulatory mechanism for dynamically titering CaMKIIactivity. These studies show that MsrA is essential for reversing CaMKIIoxidation in myocardium in vivo and that increased methionine oxidationworsens important clinical outcomes after myocardial infarction.

Structural heart disease is one of the largest public health problems inthe developed world (Jessup and Brozena, 2003). AngII and βAR receptorantagonist drugs have significantly reduced mortality in patients withstructural heart disease (Pfeffer e.g., 1992; Pfeffer e.g., 2003), andrepresent a remarkable success story for translating basic scientificunderstanding of cellular signaling into effective treatments for humandisease. Increased cardiomyocyte apoptosis appears to be an importantfeature of advanced structural heart disease (Olivetti e.g., 1997).CaMKII is activated downstream to βAR receptor stimulation (Zhang e.g.,2005) by increased Ca²⁺ _(i) (Zhu e.g., 2003). CaMKII inhibition reducesapoptosis (Zhu e.g., 2003; Yang e.g., 2006), and improves mortality(Khoo e.g., 2006) in structural heart disease models. These findingshave contributed to a growing perception that CaMKII inhibition may be anovel therapeutic strategy for treating heart failure and arrhythmias(Bers, 2005). These data reveal the importance of M281/282 oxidation forCaMKII activation and thereby provide a new molecular mechanism forunderstanding the effects of AngII in cardiomyocytes and in structuralheart disease. These present findings appear to increase the potentialimportance of CaMKII in structural heart disease by positioning CaMKIIas a critical downstream nodal signal for enhancing cardiomyocyte deathin response to excessive catecholamines, AngII and ROS. CaMKII hasproven to be a remarkably versatile signaling molecule and the recentlyrecognized role of ROS in activating CaMKII provides a new way ofunderstanding the potential for oxidant stress to engage physiologicaland disease pathways in excitable cells.

F. References for Example I

-   1. Backs, J, Song. K., Bezprozvannaya, S., Chang, S., and    Olson, E. N. (2006) CaM kinase II selectively signals to histone    deacetylase 4 during cardiomyocyte hypertrophy. J. Clin. Invest 116,    1853-1864.-   2. Barford, D (2004) The role of cysteine residues as    redox-sensitive regulatory switches. Curr. Opin. Struct. Biol. 14,    679-686.-   3. Bernstein, E., Caudy, A. A., Hammond, S. M., and    Hannon, G. J. (2001) Role for a bidentate ribonuclease in the    initiation step of RNA interference. Nature 409363-366.-   4. Bers, D. M. (2005) Beyond beta blockers. Nat. Med. 11, 379-380.-   5. Brummelkamp, T. R., Bernards, R., and Agami, R. (2002) A system    for stable expression of short interfering RNAs in mammalian cells.    Science 296, 550-553.-   6. Colbran, R. J. (1993) Inactivation of Ca²⁺/calmodulin-dependent    protein kinase II by basal autophosphorylation. J. Biol. Chem. 268,    7163-7170.-   7. Cross, J. V. and Templeton, D. J. (2004) Oxidative stress    inhibits MEKK1 by site-specific glutathionylation in the ATP-binding    domain. Biochem. J. 381, 675-683.-   8. Doerries, C. Grote, K., Hilfiker-Kleiner, D., Luchtefeld, M.,    Schaefer, A., Holland, S. M., Sorrentino, S., Manes, C., Schieffer,    B., Drexler, H., and Landmesser, U. (2007) Critical role of the    NAD(P)H oxidase subunit p47phox for left ventricular    remodeling/dysfunction and survival after myocardial infarction.    Circ. Res. 100, 894-903.-   9. Engers, R., Springer, E., Kehren. V., Simic, T., Young, D. A.,    Beier, J., Klotz, L. O., Clark, I. M., Sies, H., and    Gabbert, H. E. (2006) Rac upregulates tissue inhibitor of    metalloproteinase-1 expression by redox-dependent activation of    extracellular signal-regulated kinase signaling. FEBS J. 275,    4754-4769.-   10. Grueter, C. E., Abiria, S. A., Dzhura, I., Wu, Y., Ham, A. J.,    Mohler, P. J., Anderson, M. E., and Colbran, R. J. (2006) L-Type    Ca²⁺ Channel Facilitation Mediated by Phosphorylation of the [beta]    Subunit by CaMKII. Mol. Cell 23, 641-650.-   11. Hammond, S. M., Bernstein, E., Beach, D., and    Hannon, G. J. (2000) An RNA-directed nuclease mediates    post-transcriptional gene silencing in Drosophila cells. Nature 404,    293-296.-   12. Hare, J. M. (2001) Oxidative stress and apoptosis in heart    failure progression. Circ. Res. 89, 198-200.-   13. Hoshi, T. and Heinemann, S. H. (2001) Regulation of cell    function by methionine oxidation and reduction, J. Physiol. 531.1,    1-11,-   14. Howe, C. J., Lahair, M. M., McCubrey, J. A., and Franklin, R.    A, (2004) Redox regulation of the calcium/calmodulin-dependent    protein kinases. J Biol. Chem. 279, 44573-44581.-   15. Hudmon, A. and Schulman, H. (2002) Structure-function of the    multifunctional Ca²⁺/calmodulin-dependent protein kinase II.    Biochem. J 364, 593-611.-   16. Jessup, M. and Brozena, S. (2003) Heart failure. N. Engl. J.    Med. 348, 2007-2018.-   17. Khoo, M. S., Li, J., Singh, M. V., Yang, Y., Kannankeril, P.,    Wu, Y., Grueter, C. E., Guan, X., Oddis, C. V., Zhang, R.,    e.g. (2006) Death, cardiac dysfunction, and arrhythmias are    increased by calmodulin kinase II in calcineurin cardiomyopathy.    Circ. 114, 1352-1359.-   18. Kinugawa, S., Tsutsui, H., Hayashidani, S., Ide, T., Suematsu,    N., Satoh, S., Utsumi, H., and Takeshita, A. (2000) Treatment with    dimethylthiourea prevents left ventricular remodeling and failure    after experimental myocardial infarction in mice: role of oxidative    stress. Circ. Res. 87, 392-398.-   19. Kryukov, G. V., Kumar, R. A., Koc, A., Sun, Z., and    Gladyshev, V. N. (2002) Selenoprotein R is a zinc-containing    stereo-specific methionine sulfoxide reductase. Proc. Natl. Acad.    Sci 99, 4245-4250.-   20. Liu. H., Sadygov, R. G., and Yates, J. R., III (2004) A model    for random sampling and estimation of relative protein abundance in    shotgun proteomics. Anal. Chem. 76, 4193-4201.-   21. Lou, L. L., Lloyd, S. J., and Schulman, H. (1986) Activation of    the multifunctional Ca²⁺/calmodulin-dependent protein kinase by    autophosphorylation; ATP modulates production of an autonomous    enzyme. Proc. Nat. Acad. Sci. 83, 9497-9501. Lyle, A. N. and    Griendling, K. K. (2006) Modulation of vascular smooth muscle    signaling by reactive oxygen species. Physiology 21, 269-280.-   22. Maack, C., Kartes, T., Kilter, H., Schafers, H. J., Nickenig,    G., Bohm, M., and Laufs, U. (2003) Oxygen free radical release in    human failing myocardium is associated with increased activity of    rac1-GTPase and represents a target for statin treatment Circ. 108,    1567-1574.-   23. Meyer, T., Hanson, P. I., Stryer, L., and Schulman, H. (1992)    Calmodulin trapping by calcium-calmodulin-dependent protein kinase.    Science 256, 1199-1202.-   24. Mohler, P. J., Le Scouarnec, S., Denjoy, I., Lowe, J. S.,    Guicheney, P., Caron, L., Driskell, I. M., Schott, J. J., Norris,    K., Leenhardt, A., e.g. (2007) Defining the cellular phenotype of    “ankyrin-B syndrome” variants: human ANK2 variants associated with    clinical phenotypes display a spectrum of activities in    cardiomyocytes. Circ. 115, 432-441.-   25. Moskovitz, J., Bar-Noy, S., Williams, W. M., Requena, J.,    Berlett, B. S., and Stadtman, E. R. (2001) Methionine sulfoxide    reductase (MsrA) is a regulator of antioxidant defense and lifespan    in mammals. Proc. Natl. Acad. Sci. 98, 12920-12925.-   26. Munzel, T. and Keaney, J. F., Jr. (2001) Are ACE inhibitors a    “magic bullet” against oxidative stress? Circ. 104, 1571-1574.-   27. Olivetti, G., Abbi, R., Quaini, F., Kajstura, J., Cheng, W.,    Nitahara, J. A., Quaini, E., Di, L. C., Beltrami, C. A., Krajewski,    S., e.g. (1997) Apoptosis in the failing human heart. N. Engl. J    Med. 336, 1131-1141.-   28. Patton, B. L.; Miller, S. G., and Kennedy, M. B. (1990)    Activation of type II calcium/calmodulin-dependent protein kinase by    Ca²⁺/calmodulin is inhibited by autophosphorylation of threonine    within the calmodulin-binding domain. J. Biol. Chem. 265,    11204-11212.-   29. Pfeffer, M. A., Braunwald, E., Moye, L. A., Basta, L., Brown, E.    J., Jr., Cuddy, T. E., Davis, B. R., Geltman, E. M., Goldman, S.,    e.g. (1992) Effect of captopril on mortality and morbidity    inpatients with left ventricular dysfunction after myocardial    infarction. Results of the survival and ventricular enlargement    trial. The SAVE Investigators. N. Engl. J Med. 527, 669-677.-   30. Pfeffer, M. A., McMurray, J. J., Velazquez, E. J., Rouleau, J.    L., Kober, L., Maggioni, A. P., Solomon, S. D., Swedberg, K., Van    de, W. F., White, H., e.g. (2003) Valsartan, captopril, or both in    myocardial infarction complicated by heart failure, left ventricular    dysfunction, or both. N. Engl. J Med, 349, 1893-1906.-   31. Reynaert, N. L., van, D., V, Guala, A. S., McGovern, T.,    Hristova, M., Pantano, C., Heintz, N. H., Heim, J., Ho, Y. S.,    Matthews, D. E., Wouters, E. P., and Janssen-Heininger, Y. M. (2006)    Dynamic redox control of NF-kappaB through glutaredoxin-regulated    S-glutathionylation of inhibitory kappaB kinase beta. Proc. Natl.    Acad. Sci. 103, 13086-13091.-   32. Rosenberg, O. S., Deindl, S., Sung, R. J., Nairn, A. C, and    Kuriyan, J. (2005) Structure of the autoinhibited kinase domain of    CaMKII and SAXS analysis of the holoenzyme. Cell 123, 849-860.-   33. Ruan, H., Tang, X. D., Chen, M. L., Joiner, M. L., Sun, G.,    Brot, N., Weissbach, H., Heinemann, S. H., Iverson, I., Wu, C. F.,    and Hoshi, T. (2002) High-quality life extension by the enzyme    peptide methionine sulfoxide reductase. Proc. Natl. Acad. Sci. 99,    2748-2753.-   34. Saitoh, T. and Schwartz, J. H. (1985) Phosphorylation-dependent    subcellular translocation of a Ca²⁺/calmodulin-dependent protein    kinase produces an autonomous enzyme in Aplysia neurons. J. Cell    Biol. 100, 835-842.-   35. Santarelli, L. C., Wassef, R., Heinemann, S. H., and    Hoshi, T. (2006) Three methionine residues located within the    regulator of conductance for K⁺ (RCK) domains confer oxidative    sensitivity to large-conductance Ca²⁺-activated K⁺ channels. J.    Physiol 571, 329-348.-   36. Schulman, H. and Greengard, P. (1978) Ca²⁺-dependent protein    phosphorylation system in membranes from various tissues, and its    activation by “calcium-dependent regulator”. Proc. Nat. Acad. Sci.    75, 5432-5436.-   37. Sharma, R. V., Chapleau, M. W., Hajduczok, G., Wachtel, R. E.,    Waite, L. J., Bhalla, R. C., and Abboud, F. M. (1995) Mechanical    stimulation increases intracellular calcium concentration in nodose    sensory neurons. Neuroscience 66, 433-441.-   38. Tojo, A., Onozato, M. L., Kobayashi, N., Goto, A., Matsuoka, H.,    and Fujita, T. (2002) Angiotensin II and oxidative stress in Dahl    Salt-sensitive rat with heart failure. Hypertension 40, 834-839.-   39. Tonks, N. K. (2006) Protein tyrosine phosphatases: from genes,    to function, to disease. Nat. Rev. Mol. Cell Biol. 7, 833-846.-   40. Weiss, R. M., Ohashi, M., Miller, J. D., Young, S. G., and    Heistad, D. D. (2006) Calcific aortic valve stenosis in old    hypercholesterolemic mice. Circ. 114, 2065-2069.-   41. Weissbach, H., Etienne, F., Hoshi, T., Heinemann, S. H.,    Lowther, W. T., Matthews, B., St John, G., Nathan, C., and    Brot, N. (2002) Peptide methionine sulfoxide reductase: structure,    mechanism of action, and biological function. Arch. Biochem.    Biophys. 397, 172-178.-   42. Wu, Y., Temple, J., Zhang, R., Dzhura, I., Zhang, W.,    Trimble, R. W., Roden, D. M., Passier, R., Olson, E. N., Colbran, R.    J., and Anderson, M. E. (2002) Calmodulin kinase II and arrhythmias    in a mouse model of cardiac hypertrophy, Circ. 106, 1288-1293.-   43. Yang, Y., Zhu, W. Z., Joiner, M. L., Zhang, R., Oddis, C. V.,    Hou, Y., Yang, J., Price, E. E., Gleaves, L., Eren, M., e.g. (2006)    Calmodulin kinase II inhibition protects against myocardial cell    apoptosis in vivo. Am. J. Physiol Heart Circ. Physiol 291,    H3065-H3075.-   44. Zangerle, L., Cuenod, M., Winterhalter, K. H., and    Do, K. Q. (1992) Screening of thiol compounds:    depolarization-induced release of glutathione and cysteine from rat    brain slices. J. Neurochem. 59, 181-189.-   45. Zhabotinsky, A. M. (2000) Bistability in the    Ca²⁺/calmodulin-dependent protein kinase-phosphatase system. Biophy    J 79, 2211-2221.-   46. Zhang, R., Khoo, M. S., Wu, Y., Yang, Y., Grueter, C. E., Ni,    G., Price, E. E., Thiel, W., Guatimosim, S., Song, L. S.,    e.g. (2005) Calmodulin kinase II Inhibition protects against    structural heart disease. Nat. Med. 111, 409-417.-   47. Zhu, W. Z., Wang, S. Q., Chakir, K. Yang, D. M., Zhang, T.,    Brown, J. H., Devic, E., Kobilka, B. K., Cheng, H. P., and    Xiao, R. P. (2003) Linkage of beta(1)-adrenergic stimulation to    apoptotic heart cell death through protein kinase A-independent    activation of Ca²⁺/calmodulin kinase II. J. of Clin. Inv. 111,    617-625.-   48. Zhu, W. Z., Woo, A. Y., Yang, D. M., Cheng, H., Crow, M. T., and    Xiao, R. P. (2007) Activation of CaMKII is a common intermediate of    diverse death stimuli-induced heart muscle cell apoptosis. J. of    Biol. Chem. 282, 10833-10839.-   49. Zimmerman, M. C., Dunlay, R. P., Lazartigues, E., Zhang, Y.,    Sharma, R. V., Engelhardt, J. F., and Davisson, R. L. (2004)    Requirement for Rac1-dependent NADPH oxidase in the cardiovascular    and dipsogenic actions of angiotensin II in the brain. Circ. Res.    95, 532-539.

Example II Characterization of Antiserum Against oxCaMKII

Rabbit Antiserum Against Oxidized Calcium/Calmodulin Dependent Kinase IIwas prepared as described in Example I and tested as follows.

A. Immunodetection of Oxidized CaMKII in Heart Tissues

To test the specificity of this antiserum, Western blots of isolatedCaMKII after no treatment, oxidation with H₂O₂, or phosphorylation withATP were performed. CaMKII samples were probed for either total proteinusing a general CaMKII antibody or for oxidized CaMKII withanti-oxCaMKII antiserum. These results demonstrate the specificity ofthis antiserum for the oxidized form of CaMKII. (See FIG. 14A). To testthe efficacy of the antiserum in tissue staining, mice were treated witheither saline or angiotensin II, an oxidant stimulating agent in cardiactissue, and stained heart sections and performed immunofluorescentstains for oxidized CaMKII. The heart treated with angiotensin II showedsignificant staining relative to the control. (See FIG. 14B).

B. Angiotensin II (AngII) and Endotoxin (Endo) Treatment Results inIncreased Oxidized CaMKII (oxCaMKII) in Blood

In order to test whether the anti-oxCaMKII antiserum was suitable fordetecting increased CaMKII oxidation in blood under disease conditionsassociated with enhanced oxidative stress, validated mouse models wereutilized. Wild type (WT) and MsrA−/− mice were treated with saline,AngII, or Endo and sacrificed 6 hours later. (See FIG. 15). Western blotof blood samples and ImageJ analysis were used to quantify total andoxidized CaMKII. (See FIG. 15). Immunoblots of peripheral blood showincreased oxidized CaMKII in heart failure, sepsis and premature agingdisease models, (See FIG. 15).

C. Cancer and 2DG Treatment are Associated with Concomitant Increases inoxCaMKII in Blood

Mice were exposed to cancer cells and treated with either saline or 2DG.Those mice that tailed to develop tumors after 60 days were labeled ascontrols. (See FIG. 16). Western blot of blood samples and ImageJanalysis were used to quantify total and oxidized CaMKII. (See FIG. 16).Immunoblots of peripheral blood showed increased oxidized CaMKII inhealthy mice and mice with cancer. (See FIG. 16).

D. Detection of oxCAMKII in Mice Bearing Cancer

Mice with no cancer, FADU (head and neck cancer), or human lung cancerwere treated with saline, 2DG, radiation (rad), or given a ketogenicdiet (keto). Blood samples were collected from the mice and oxidizedCaMKII and total CaMKII were measured by immunoassay. (See FIG. 17.)

E. Cancer and Varied Treatments Increase Blood oxCaMKII in an AdditiveManner

Mice were divided into four groups based on the presence or absence ofcancer and whether or not they received treatment. Western blot of bloodsamples and ImageJ analysis were used to quantify total and oxidizedCaMKII. (See FIG. 18). Both cancer and varied treatments increase bloodoxCaMKII in an additive manner.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein. The terms and expressions whichhave been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention. Thus, itshould be understood that although the present invention has beenillustrated by specific embodiments and optional features, modificationand/or variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member, any subgroup of members of theMarkush group or other group, or the totality of members of the Markushgroup or other group.

Citations to a number of patent and non-patent references are madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

We claim:
 1. A method of detecting oxidized calcium/calmodulin-dependentprotein kinase II (oxCaMKII), the method comprising: (a) contacting abiological sample from a patient with a composition comprising apurified monoclonal antibody or antigen-binding fragment thereof thatbinds specifically to oxCaMKII to form a complex, wherein the monoclonalantibody or antigen-binding fragment binds specifically to oxCaMKIIdelta having an amino acid sequence comprising SEQ ID NO:12 and havingoxidized methionine residues at positions 281 and 282, and wherein themonoclonal antibody or antigen-binding fragment does not bind to CaMKIIdelta having an amino acid sequence comprising SEQ ID NO:12 and havingnon-oxidized methionine residues at positions 281 and 282; and (b)detecting the complex.
 2. The method of claim 1, wherein the biologicalsample is blood or plasma.
 3. The method of claim 1, further comprising:(c) characterizing cardiac disease in the patient.
 4. The method ofclaim 1, wherein the patient has or is at risk for developing a diseaseor condition selected from a group consisting of cardiac disease,cancer, premature aging, atherosclerosis, Alzheimer's disease, orsepsis, and the method further comprises: (c) administering atherapeutic agent based on detecting or not detecting oxCaMKII.
 5. Themethod of claim 1, wherein the patient is undergoing therapy with anangiotensin converting enzyme (ACE) inhibitor and the method furthercomprises: (c) modulating the therapy based on detecting or notdetecting oxCaMKII.
 6. The method of claim 1, wherein modulating thetherapy comprises increasing dosage of the ACE inhibitor.
 7. The methodof claim 6, wherein modulating the therapy comprises decreasing dosageof the ACE inhibitor or ceasing the therapy.
 8. The method of claim 1,wherein the patient is undergoing anti-cancer therapy and the methodfurther comprises: (c) modulating the therapy based on detecting or notdetecting oxCaMKII.
 9. The method of claim 8, wherein the anti-cancertherapy is selected from a group consisting of radiation therapy,chemotherapy, nutritional therapy, and combinations thereof.